Expression of xenogenous (human) immunoglobulins in cloned, transgenic ungulates

ABSTRACT

The present invention relates to the production of a transgenic bovine which comprises a genetic modification that results in inactivation and loss of expression of its endogenous antibodies, and the expression of xenogenous antibodies, preferably human antibodies. This is effected by inactivation of the IgM heavy chain expression and, optionally, by inactivation of the Ig light chain expression, and by the further introduction of an artificial chromosome which results in the expression of non-bovine antibodies, preferably human antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the filing date of U.S.provisional patent application No. 60/311,625, filed Aug. 9, 2001 andU.S. provisional patent application No. 60/256,458, filed Dec. 20, 2000,and is a continuation-in-part of U.S. utility application Ser. No.09/714,185, filed Nov. 17, 2000.

FIELD OF THE INVENTION

[0002] The present invention is a genetically modified ungulate thatcontains either part or all of a xenogenous antibody gene locus, whichundergoes rearrangement and expresses a diverse population of antibodymolecules. In particular, the xenogenous antibody gene may be of humanorigin. In addition, the present invention provides for an ungulate inwhich expression of the endogenous antibody genes is either reduced oreliminated. The genetic modifications in the ungulate (for example,bovine) are made using a combination of nuclear transfer and moleculartechniques. These cloned, transgenic ungulates provide a replenishable,theoretically infinite supply of xenogenous polyclonal antibodies,particularly human antibodies, which have use, e.g. as therapeutics,diagnostics and for purification purposes.

BACKGROUND OF THE INVENTION

[0003] In 1890, Shibasaburo Kitazato and Emil Behring reported anexperiment with extraordinary results; particularly, they demonstratedthat immunity can be transferred from one animal to another by takingserum from an immune animal and injecting it into a non-immune one. Thislandmark experiment laid the foundation for the introduction of passiveimmunization into clinical practice. Today, the preparation and use ofhuman immunoglobulin (Ig) for passive immunization is standard medicalpractice. In the United States alone, there is a $1,400,000,000 perannum market for human Ig, and each year more than 16 metric tons ofhuman antibody is used for intravenous antibody therapy. Comparablelevels of consumption exist in the economies of most highlyindustrialized countries, and the demand can be expected to grow rapidlyin developing countries. Currently, human antibody for passiveimmunization is obtained from the pooled serum of human donors. Thismeans that there is an inherent limitation in the amount of humanantibody available for therapeutic and prophylactic usage. Already, thedemand exceeds the supply and severe shortfalls in availability havebeen routine.

[0004] In an effort to overcome some of the problems associated with theinadequate supply of human Ig, various technologies have been developed.For example, the production of human Ig by recombinant methods in tissueculture is routine. Particularly, the recombinant expression of human Igin CHO expression systems is well known, and is currently utilized forthe production of several human immunoglobulins (Igs) and chimericantibodies now in therapeutic use.

[0005] Mice retaining an unrearranged human immunoglobulin gene havebeen developed for the production of human antibodies (e.g., monoclonalantibodies) (see, for example, WO98/24893; WO96/33735; WO 97/13852;WO98/24884; WO97/07671(EP 0843961); U.S. Pat. No. 5,877,397; U.S. Pat.No. 5,874,299; U.S. Pat. No. 5,814,318; U.S. Pat. No. 5,789,650; U.S.Pat. No. 5,770,429; U.S. Pat. No. 5,661,016; U.S. Pat. No. 5,633,425;U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,569,825; and U.S. Pat. No.5,545,806).

[0006] Additionally, WO00/10383 (EP 1106061) describes modifying a humanchromosome fragment and transferring the fragment into certain cells viamicrocell fusion.

[0007] Further, WO01/35735 describes a bovine IgM heavy chain knockout.

[0008] U.S. Pat. No. 5,849,992 issued Dec. 15, 1998 to Meade et al., aswell as U.S. Pat. No. 5,827,690 issued Oct. 27, 1998 to Meade et al.,describe the production of monoclonal antibodies in the milk oftransgenic animals including mice, sheep, pigs, cows, and goats whereinthe transgenic animals expressed human Ig genes under the control ofpromoters that provide for the expression of the antibodies in mammaryepithelial cells. Essentially, this results in the expression of theantibodies in the milk of such animals, for example a cow.

[0009] However, notwithstanding what has been previously reported,improved methods and enhanced transgenic animals, especially cows, thatproduce antibodies (particularly, polyclonal antibodies) of desiredspecies, particularly human Igs, in the bloodstream and which produce anarray of different antibodies which are specific to a desired antigenwould be highly desirable. Most especially, the production of human Igsin ungulates, such as cows, would be particularly beneficial given that(1) cows could produce large quantities of antibody, (2) cows could beimmunized with human or other pathogens and (3) cows could be used tomake human antibodies against human antigens. The availability of largequantities of polyclonal antibodies would be advantageous for treatmentand prophylaxis for infectious disease, modulation of the immune system,removal of human cells, such as cancer cells, and modulation of specifichuman molecules. While human Ig has been expressed in mice, it isunpredictable whether human Ig will be fractionally rearranged andexpressed in bovines, or other ungulates, because of differences inantibody gene structure, antibody production mechanism, and B cellfunction. In particular, unlike mice, cattle and sheep differ fromhumans in their immunophysiology (Lucier et al., J. Immunol. 161: 5438,1998; Pamg et al., J. Immunol. 157:5478, 1996; and Butler, Rev. Sci.Tech. 17:43, 2000). For example antibody gene diversification in bovinesand ovines relies much more on gene conversion than gene rearrangementas in humans and mice. Also, the primary location of B cells in humansand mice is in the bone marrow, whereas in bovines and ovines B cellsare located in the illeal Peyer's patch. Consequently, it would havebeen difficult, if not impossible, prior to the present invention, topredict whether immunoglobulin rearrangement and diversification of ahuman immunoglobulin loci would take place within the bovine (or otherungulate) B cell lineage. In addition, it would also have beenunpredictable whether a bovine would be able to survive, i.e., elicitits normal immune functions, in the absence of its endogenous Ig or withinterference from human antibodies. For example, it is not certain ifbovine B cells expressing human Ig would correctly migrate to the illealPeyer's Patch in bovines because this does not happen in humans. Also,it is not clear if human Fc receptor function; which mediates complementactivation, induction of cytokine release, and antigen removal; would benormal in a bovine system.

BRIEF DESCRIPTION OF THE INVENTION

[0010] It is an object of the invention to produce a transgenic ungulate(for example, a transgenic bovine) that rearranges and expresses ahuman, or other species Ig gene locus. Preferably, this is accomplishedby stably introducing a human chromosome fragment containing human Iggenes, in order to produce a transgenic ungulate (for example, bovine)having B cells that produce human or another species Ig, in addition toor in lieu of endogenous Igs. This may also be accomplished byintegrating a nucleic acid encoding a xenogenous immunoglobulin chain orxenogenous antibody into a chromosome of an ungulate. It is a furtherobject of the present invention to produce transgenic ungulates (forexample, transgenic bovines) wherein the expression of endogenous Ig hasbeen reduced or knocked out. For example, a nonsense or deletionmutation may be introduced into a nucleic acid encoding an endogenousimmunoglobulin chain or antibody.

[0011] It is a more specific object of the invention to produce atransgenic ungulate (for example, a transgenic bovine) wherein theconstant region exon of the light chain loci and/or the mu constantregions exons have been knocked out, and an artificial chromosomecontaining a gene locus encoding another species' immunoglobulin,preferably human, has been stably incorporated.

[0012] It is a more specific object of the invention to produce a clonedungulate (for example, a cloned bovine) by the use of nuclear transferand homologous recombination procedures wherein the endogenous constantregion exon of the light chain loci and/or the mu constant region exonsof the heavy chain locus have been knocked out, and an artificialchromosome(s) comprising xenogenous heavy and light chain Ig genes,preferably a human artificial chromosome(s) containing human heavy andlight chain Ig loci, has been stably introduced, resulting in atransgenic ungulate which produces Ig of another species, preferablyhuman, and which does not produce its endogenous Ig.

[0013] It is another object of the invention to produce an ungulate (forexample, a bovine) somatic or embryonic stem (ES) cell, preferably afibroblast or B cell, and more preferably a male somatic cell, whereinone or both alleles of the endogenous IgM heavy chain gene has beenmutated, for example, disrupted by homologous recombination. It is arelated object of the invention to produce a cloned ungulate (forexample, bovine) fetus and offspring wherein one or both alleles of theIgM heavy chain gene locus has been mutated, for example, disrupted byhomologous recombination.

[0014] It is still another object of the invention to produce anungulate (for example, a bovine) somatic or ES cell, preferably afibroblast or B cell, e.g., a female or male somatic cell, wherein oneallele of the IgM heavy chain gene has been mutated, for example,disrupted by homologous recombination.

[0015] It is a related object of the invention to produce a cloned(ungulate, for example, bovine) fetus or offspring wherein one allele ofthe endogenous heavy chain IgM gene has been mutated, for example,disrupted by homologous recombination.

[0016] It is still another object of the invention to produce male andfemale heavy and light chain hemizygous knockout (M and F Hemi H/L)fetuses and ungulate calves by mating male and female ungulates (forexample, bovines) which respectively contain a mutation, for example, adisruption of one allele of the endogenous IgM or a disruption of oneallele of an Ig light chain or by sequential homologous recombination.

[0017] It is still another object of the invention to produce ahomozygous knockout (Homo H/L) fetus wherein both heavy chain alleles ofthe IgM gene have been disrupted and both alleles of the Ig light chainhave been disrupted by sequential homologous recombination or by matingof the aforementioned male and female heavy and light chain hemizygousknockouts (M and F Hemi H/L).

[0018] It is another specific object of the invention to insert anucleic acid (for example, an artificial chromosome) that contains genesnecessary for the functional expression of non-ungulate Igs or theirheavy or light chains. Preferably, these Igs are human Igs produced byintroduction of nucleic acid encoding these Igs or Ig chains into a Homoor a Hemi H/L ungulate (for example, bovine) somatic cell, preferably afibroblast, and producing cloned ungulates (for example, cloned bovines)wherein the nucleic acid (for example, human artificial chromosome DNA)is transmitted into the germ line.

[0019] It is still another object of the invention to introduce anartificial chromosome, preferably a human artificial chromosome (HAC),that contains genes that provide for Ig expression into theaforementioned homozygous knockout (Homo H/L) cells and generateungulates (for example, cattle) by nuclear transfer which expressnon-ungulate Igs, preferably human Igs, in response to immunization andwhich undergo affinity maturation.

[0020] As used herein, by “artificial chromosome” is meant a mammalianchromosome or fragment thereof which has an artificial modification suchas the addition of a selectable marker, the addition of a cloning site,the deletion of one or more nucleotides, the substitution of one or morenucleotides, and the like. By “human artificial chromosome (HAC)” ismeant an artificial chromosome generated from one or more humanchromosome(s). An artificial chromosome can be maintained in the hostcell independently from the endogenous chromosomes of the host cell. Inthis case, the HAC can stably replicate and segregate along sideendogenous chromosomes. Alternatively, it may be translocated to, orinserted into, an endogenous chromosome of the host cell. Two or moreartificial chromosomes can be introduced to the host cell simultaneouslyor sequentially. For example, artificial chromosomes derived from humanchromosome #14 (comprising the Ig heavy chain gene), human chromosome #2(comprising the Ig kappa chain gene), and human chromosome #22(comprising the Ig lambda chain gene) can be introduced. Alternatively,an artificial chromosome(s) comprising both a xenogenous Ig heavy chaingene and Ig light chain gene, such as ΔHAC or ΔHAC, may be introduced.Preferably, the heavy chain loci and the light chain loci are ondifferent chromosome arms (i.e., on different side of the centromere).In still other preferred embodiments, the total size of the HAC is lessthan or equal to approximately 10, 9, 8, or 7 megabases.

[0021] It is still another object of the invention to provide a sourceof human or other Ig for passive immunization derived from a transgenicungulate (for example, a transgenic bovine) that contains and expressesIg genes carried on an introduced nucleic acid (for example, anartificial chromosome, and preferably a human artificial chromosome(HAC)) containing human Ig heavy and light chain genes. In the presentinvention, these nucleic acids (for example, HACs) include naturallyarranged segments of human chromosomes (human chromosomal fragments) orartificial chromosomes that comprise artificially engineered humanchromosome fragments, i.e., they may be rearranged relative to the humangenome.

[0022] It is yet another object of the invention to produce hybridomasand monoclonal antibodies using B cells derived from the above-describedtransgenic ungulates (for example, transgenic bovines).

[0023] It is still another object of the invention to produce ungulateantiserum or milk that includes polyclonal human Ig. Such human Ig,preferably human IgG, may be used as intravenenous immunoglobulin (IVIG)for the treatment or prevention of disease in humans. The polyclonalhuman Ig are preferably reactive against an antigen of interest.

[0024] It is yet another object of the invention to produce a transgenicungulate with one or more mutations in an endogenous gene or genes. Thetransgenic ungulate is produced by inserting a cell, a chromatin massfrom a cell, or a nucleus from a cell into an oocyte. The cell has afirst mutation in an endogenous gene that is not naturally expressed bythe cell. The oocyte or an embryo formed from the oocyte is transferredinto the uterus of a host ungulate under conditions that allow theoocyte or the embryo to develop into a fetus. Preferably, the fetusdevelops into a viable offspring. In other preferred embodiments, thefirst mutation is introduced into the cell by inserting a nucleic acidcomprising a cassette which includes a promoter operably linked to anucleic acid encoding a selectable marker and operably linked to one ormore nucleic acids having substantial sequence identity to theendogenous gene to be mutated, whereby the cassette is integrated intoone endogenous allele of the gene. In other preferred embodiments, themutation is introduced in the cell by inserting into the cell a nucleicacid comprising a first cassette which includes a first promoteroperably linked to a nucleic acid encoding a first selectable marker andoperably linked to a first nucleic acid having substantial sequenceidentity to the endogenous gene to be mutated, whereby the firstcassette is integrated into a first endogenous allele of the geneproducing a first transgenic cell. Into the first transgenic cell isinserted a nucleic acid comprising a second cassette which includes asecond promoter operably linked to a nucleic acid encoding a secondselectable marker and operably linked to a second nucleic acid havingsubstantial sequence identity to the gene. The second selectable markerdiffers from the first selectable marker, and the second cassette isintegrated into a second endogenous allele of the gene producing asecond transgenic cell. In still other preferred embodiments, a cell isisolated from the embryo, the fetus, or an offspring produced from thefetus, and another mutation is introduced into a gene of the cell. Asecond round of nuclear transfer is then performed using the resultingcell, a chromatin mass from the cell, or a nucleus from the cell toproduce a transgenic ungulate with two or more mutations. The mutationsare in the same or different alleles of a gene or are in differentgenes. In preferred embodiments, the cell that is mutated is afibroblast (e.g., a fetal fibroblast). Preferably, the endogenous genethat is mutated is operably linked to an endogenous promoter that is notactive in a fibroblast. In other preferred embodiments, the endogenouspromoter operably linked to the endogenous gene that is mutated is lessthan 80, 70, 60, 50, 40, 30, 20, 10% as active as an endogenous promoteroperably linked to a endogenous housekeeping gene such as GAPDH.Promoter activity may be measured using any standard assay, such asassays that measure the level of mRNA or protein encoded by the gene(see, for example, Ausubel et al. Current Protocols in MolecularBiology, volume 2, p. 11.13.1-11.13.3, John Wiley & Sons, 1995). Thismethod for generating a transgenic ungulate has the advantage ofallowing a gene that is not expressed in the donor cell (i.e., the cellthat is the source of the genetic material used for nuclear transfer) tobe mutated.

[0025] Accordingly, the invention as claimed features a transgenicungulate having one or more nucleic acids encoding all or part of axenogenous immunoglobulin (Ig) gene which undergoes rearrangement andexpresses more than one xenogenous Ig molecule. In a preferredembodiment, the nucleic acid encoding all or part of a xenogenous Iggene is substantially human. Preferably, the nucleic acid encodes anxenogenous antibody, such as a human antibody or a polyclonal antibody.In various embodiments, the Ig chain or antibody is expressed in serumand/or milk. In other embodiments, the nucleic acid is contained withina chromosome fragment, such as a ΔHAC or a ΔΔHAC. In yet otherembodiments, the nucleic acid is maintained in an ungulate cellindependently from the host chromosome.

[0026] In still other embodiments, the nucleic acid is integrated into achromosome of the ungulate. In yet other embodiments, the nucleic acidincludes unrearranged antibody light chain nucleic acid segments inwhich all of the nucleic acid segments encoding a V gene segment areseparated from all of the nucleic acid segments encoding a J genesegment by one or more nucleotides. In yet other embodiments, thenucleic acid includes unrearranged antibody heavy chain nucleic acidsegments in which either (i) all of the nucleic acid segments encoding aV gene segment are separated from all of the nucleic acid segmentsencoding a D gene segment by one or more nucleotides and/or (ii) all ofthe nucleic acid segments encoding a D gene segment are separated fromall of the nucleic acid segments encoding a J gene segment by one ormore nucleotides. In yet another preferred embodiment, the ungulate hasa mutation in one or both alleles of an endogenous Ig gene,alpha-(1,3)-galactosyltransferase gene, prion gene, and/or J chain gene.In other preferred embodiments, the ungulate has a nucleic acid encodingan exogenous J chain, such as a human J chain. Preferably, the ungulateis a bovine, ovine, porcine, or caprine.

[0027] In another aspect, the invention features a transgenic ungulatehaving a mutation that reduces the expression of an endogenous antibody.Preferably, the mutation reduces the expression of functional IgM heavychain or substantially eliminates the expression of functional IgM heavychain. In other preferred embodiments, the mutation reduces theexpression of functional Ig light chain or substantially eliminates theexpression of functional Ig light chain. In yet other preferredembodiments, the mutation reduces the expression of functional IgM heavychain and functional Ig light chain, or the mutation substantiallyeliminates the expression of functional IgM heavy chain and functionalIg light chain. Preferably, the ungulate also has a mutation in one orboth alleles of an endogenous nucleic acid encodingalpha-(1,3)-galactosyltransferase, prion protein, and/or J chain. Inother preferred embodiments, the ungulate has a nucleic acid encoding anexogenous J chain, such as a human J chain. In another preferredembodiment, the ungulate has one or more nucleic acids encoding all orpart of a xenogenous Ig gene which undergoes rearrangement and expressesmore than one xenogenous Ig molecule. Preferably, the nucleic acidencoding all or part of a xenogenous Ig gene is substantially human. Inother preferred embodiments, the nucleic acid encodes a xenogenousantibody, such as a an antibody from another genus (e.g., a humanantibody) or a polyclonal antibody. In various embodiments, the Ig chainor antibody is expressed in serum. In other embodiments, the nucleicacid is contained within a chromosome fragment, such as a ΔHAC or aΔΔHAC. In yet other embodiments, the nucleic acid is maintained in anungulate cell independently from the host chromosome. In still otherembodiments, the nucleic acid is integrated into a chromosome of theungulate. Preferably, the ungulate is a bovine, ovine, porcine, orcaprine.

[0028] The invention also provides cells obtained from any of theungulates of the invention or cells that are useful in the production ofany of the ungulates of the invention.

[0029] Accordingly, in another aspect, the invention features anungulate somatic cell having one or more nucleic acids encoding all orpart of a xenogenous Ig gene that is capable of undergoing rearrangementand expressing one or more xenogenous Ig molecules in B cells.Preferably, the nucleic acid encoding all or part of a xenogenous Iggene encodes a xenogenous antibody. In various embodiments, the nucleicacid is contained in a chromosome fragment, such as a ΔHAC or a ΔΔHAC.In yet other embodiments, the nucleic acid is maintained in an ungulatecell independently from the host chromosome. In still other embodiments,the nucleic acid is integrated into a chromosome of the cell. In anotherembodiment, the nucleic acid is substantially human. Preferably, thexenogenous antibody is an antibody from another genus, such as a humanantibody. Preferably, the cell has a mutation in one or both alleles ofan endogenous nucleic acid encoding alpha-(1,3)-galactosyltransferase,prion protein, and/or J chain. In other preferred embodiments, the cellhas a nucleic acid encoding an exogenous J chain, such as a human Jchain. Exemplary ungulate cells include fetal fibroblasts and B-cells.Preferably, the ungulate is a bovine, ovine, porcine, or caprine.

[0030] In another aspect, the invention features an ungulate somaticcell having a mutation in a nucleic acid encoding an Ig heavy and/orlight chain. In preferred embodiments, the cell has a mutation in one orboth alleles of the IgM heavy chain or the Ig light chain. Exemplarymutations include nonsense and deletion mutations. Preferably, the cellhas a mutation in one or both alleles of an endogenous nucleic acidencoding alpha-(1,3)-galactosyltransferase, prion protein, and/or Jchain. In other preferred embodiments, the cell has a nucleic acidencoding an exogenous J chain, such as a human J chain. In preferredembodiments, the cell also has one or more nucleic acids encoding all orpart of a xenogenous Ig gene that is capable of undergoing rearrangementand expressing one or more xenogenous Ig molecules in B cells.Preferably, the nucleic acids encoding all or part of a xenogenous Iggene is substantially human and/or encodes a xenogenous antibody, suchas an antibody from another genus (e.g., a human antibody). In variousembodiments, the nucleic acid is contained in a chromosome fragment,whereby the nucleic acid is maintained in the ungulate cellindependently from the host chromosome. Preferred chromosome fragmentsinclude ΔHAC and ΔΔHAC. In yet other embodiments, the nucleic acid ismaintained in an ungulate cell independently from the host chromosome.In still other embodiments, the nucleic acid is integrated into achromosome of the cell. Exemplary ungulate cells include fetalfibroblasts and B-cells. Preferably, the ungulate is a bovine, ovine,porcine, or caprine.

[0031] In another aspect, the invention features a hybridoma formed fromthe fusion of an ungulate B-cell of the invention with a myeloma cell.Preferably, the hybridoma secretes an exogenous antibody, such as ahuman antibody.

[0032] The invention also provides methods for producing antibodiesusing an ungulate of the invention. One such method involvesadministering one or more antigens of interest to an ungulate having oneor more nucleic acids encoding a xenogenous antibody gene locus. Thenucleic acid segments in the gene locus undergo rearrangement resultingin the production of antibodies specific for the antigen, and theantibodies are recovered from the ungulate. In various embodiments, thenucleic acid encoding the xenogenous antibody gene locus is contained ina chromosome fragment, such as a ΔHAC or a ΔΔHAC. Preferably, thechromosome fragment is maintained in an ungulate cell independently fromthe host chromosome. In other embodiments, the nucleic acid isintegrated into a chromosome of the ungulate. Preferably, the nucleicacid is substantially human. In other preferred embodiments, the lightchain of the antibodies and/or the heavy chain of the antibodies isencoded by a human nucleic acid. The antibodies may be monoclonal orpolyclonal. Monoclonal and polyclonal antibodies against particularantigen have a variety of uses; for example, they may be used asingredients in prophylactic or therapeutic compositions for infection ofpathogenic microorganisms such as bacteria or viruses. In variousembodiments, the antibodies are recovered from the serum or milk of theungulate. In preferred embodiments, the ungulate has a mutation thatreduces the expression of an endogenous antibody, that reduces theexpression of functional IgM heavy chain, or that reduces the expressionof functional Ig light chain. Preferably, the ungulate has a mutation inone or both alleles of an endogenous nucleic acid encodingalpha-(1,3)-galactosyltransferase, prion protein, and/or J chain. Inother preferred embodiments, the ungulate has a nucleic acid encoding anexogenous J chain, such as a human J chain. Preferably, the ungulate isa bovine, ovine, porcine, or caprine.

[0033] In a related aspect, the invention features another method ofproducing antibodies. This method involves recovering xenogenousantibodies from an ungulate having nucleic acid encoding a xenogenousantibody gene locus. The nucleic acid segments in the gene locus undergorearrangement resulting in the production of xenogenous antibodies. Invarious embodiments, the nucleic acid encoding a xenogenous antibodygene locus is contained in a chromosome fragment, such as a ΔHAC or aΔΔHAC. In particular embodiments, the nucleic acid is maintained in anungulate cell independently from the host chromosome. In still otherembodiments, the nucleic acid is integrated into a chromosome of theungulate. Preferably, the nucleic acid is substantially human. Inparticular embodiments, the light chain of the antibodies and/or theheavy chain of the antibodies is encoded by a human nucleic acid. Theantibodies may be monoclonal or polyclonal. In particular embodiments,polyclonal antibodies, such as IgG antibodies generated withoutimmunization of the ungulate with a specific antigen, are used as atherapeutic substitute for IVIG (intraveneous immunoglobulin) producedfrom human serum. In various embodiments, the antibodies are recoveredfrom the serum or milk of the ungulate. Preferably, the ungulate has amutation that reduces the expression of an endogenous antibody, reducesthe expression of functional IgM heavy chain, or reduces the expressionof functional Ig light chain. Preferably, the ungulate has a mutation inone or both alleles of an endogenous nucleic acid encodingalpha-(1,3)-galactosyltransferase, prion protein, and/or J chain. Inother preferred embodiments, the ungulate has a nucleic acid encoding anexogenous J chain, such as a human J chain. Preferably, the ungulate isa bovine, ovine, porcine, or caprine.

[0034] The invention also provides methods for producing transgenicungulates. These methods may be used to produce transgenic ungulateshaving a desired mutation or having a desired xenogenous nucleic acid.

[0035] In one such aspect, the invention features a method of producinga transgenic ungulate that involves inserting a cell, a chromatin massfrom a cell, or a nucleus from a cell into an oocyte. The cell includesa first mutation in an endogenous antibody heavy chain and/or lightchain nucleic acid. The oocyte or an embryo formed from the oocyte istransferred into the uterus of a host ungulate under conditions thatallow the oocyte or the embryo to develop into a fetus. Preferably, thefetus develops into a viable offspring. Preferably, the cell used in theproduction of the transgenic ungulate has a mutation in one or bothalleles of an endogenous nucleic acid encodingalpha-(1,3)-galactosyltransferase, prion protein, and/or J chain. Inother preferred embodiments, the cell has a nucleic acid encoding anexogenous J chain, such as a human J chain. In other embodiments, thecell includes one or more nucleic acids encoding all or part of axenogenous Ig gene that is capable of undergoing rearrangement andexpressing one or more xenogenous Ig molecules in B cells. Preferably,the nucleic acid encoding all or part of a xenogenous Ig gene encodes axenogenous antibody. In yet other embodiments, the nucleic acid isintegrated into a chromosome of the cell. Preferably, the xenogenousantibody is an antibody from another genus, such as a human antibody. Inparticular embodiments, the nucleic acid is contained in a chromosomefragment, such as a ΔHAC or a ΔΔHAC. In other particular embodiments,the chromosome fragment is maintained in an ungulate cell independentlyfrom the host chromosome. Preferably, the ungulate is a bovine, ovine,porcine, or caprine.

[0036] In various embodiments of the above aspect, the method alsoincludes isolating a cell from the embryo, the fetus, or an offspringproduced from the fetus and introducing a second mutation in anendogenous antibody heavy chain and/or light chain nucleic acid in thecell. The cell, a chromatin mass from the cell, or a nucleus from thecell is inserted into an oocyte, and the oocyte or an embryo formed fromthe oocyte is transferred into the uterus of a host ungulate underconditions that allow the oocyte or the embryo to develop into a fetus.

[0037] In other embodiments of the above aspect, the cell used forgeneration of the transgenic ungulate is prepared by a method thatincludes inserting into the cell a nucleic acid having a cassette whichincludes a promoter operably linked to a nucleic acid encoding aselectable marker and operably linked to one or more nucleic acidshaving substantial sequence identity to the antibody heavy chain orlight chain nucleic acid. The cassette is integrated into one endogenousallele of the antibody heavy chain or light chain nucleic acid.

[0038] In other embodiments, the cell is produced by inserting into thecell a nucleic acid having a first cassette which includes a firstpromoter operably linked to a nucleic acid encoding a first selectablemarker and operably linked to a first nucleic acid having substantialsequence identity to the antibody heavy chain or light chain nucleicacid. The first cassette is integrated into a first endogenous allele ofthe antibody heavy chain or light chain nucleic acid producing a firsttransgenic cell.

[0039] Into the first transgenic cell is inserted a nucleic acid havinga second cassette which includes a second promoter operably linked to anucleic acid encoding a second selectable marker and operably linked toa second nucleic acid having substantial sequence identity to theantibody heavy chain or light chain nucleic acid. The second selectablemarker differs from the first selectable marker. The second cassette isintegrated into a second endogenous allele of the antibody heavy chainor light chain nucleic acid producing a second transgenic cell.

[0040] In yet another aspect, the invention features another method ofproducing a transgenic ungulate. This method involves inserting a cellhaving one or more xenogenous nucleic acids into an oocyte. Thexenogenous nucleic acid encodes all or part of a xenogenous Ig gene, andthe gene is capable of undergoing rearrangement and expressing more thanone xenogenous Ig molecule in B cells. The oocyte or an embryo formedfrom the oocyte is transferred into the uterus of a host ungulate underconditions that allow the oocyte or the embryo to develop into a fetus.Preferably, the fetus develops into a viable offspring. Preferably, thenucleic acid encoding all or part of a xenogenous Ig gene encodes axenogenous antibody. In other preferred embodiments, the antibody is apolyclonal antibody. In yet other preferred embodiments, theimmunogloblulin chain or antibody is expressed in serum and/or milk. Invarious embodiments, the nucleic acid is contained in a chromosomefragment, such as a ΔHAC or a ΔΔHAC. The nucleic acid can be maintainedin an ungulate cell independently from the host chromosome or integratedinto a chromosome of the cell. Preferably, the nucleic acid issubstantially human. In other embodiments, the xenogenous antibody is anantibody from another genus, such as a human antibody. Preferably, theungulate is a bovine, ovine, porcine, or caprine.

[0041] In yet another related aspect, the invention features anothermethod of producing a transgenic ungulate. This method involvesinserting a cell, a chromatin mass from a cell, or a nucleus from a cellinto an oocyte. The cell includes a first mutation in an endogenous genethat is not naturally expressed by the cell. The oocyte or an embryoformed from the oocyte is transferred into the uterus of a host ungulateunder conditions that allow the oocyte or the embryo to develop into afetus. Preferably, the fetus develops into a viable offspring.Preferably, the gene that is mutated encodes an antibody,alpha-(1,3)-galactosyltransferase, prion protein, or J chain. In anotherpreferred embodiment, the cell used in the production of the transgenicungulate is a fibroblast, such as a fetal fibroblast.

[0042] In various embodiments of the above method, the cell is preparedby inserting into the cell a nucleic acid having a cassette whichincludes a promoter operably linked to a nucleic acid encoding aselectable marker and operably linked to one or more nucleic acidshaving substantial sequence identity to the gene; whereby the cassetteis integrated into one endogenous allele of the gene. In otherembodiments, the cell is produced by inserting into the cell a nucleicacid having a first cassette which includes a first promoter operablylinked to a nucleic acid encoding a first selectable marker and operablylinked to a first nucleic acid having substantial sequence identity tothe gene. The first cassette is integrated into a first endogenousallele of the gene producing a first transgenic cell. Into the firsttransgenic cell is inserted a nucleic acid having a second cassettewhich includes a second promoter operably linked to a nucleic acidencoding a second selectable marker and operably linked to a secondnucleic acid having substantial sequence identity to the gene. Thesecond selectable marker differs from the first selectable marker. Thesecond cassette is integrated into a second endogenous allele of thegene producing a second transgenic cell.

[0043] In other embodiments of the above aspect, the method alsoincludes introducing a second mutation into the transgenic ungulate. Inthese embodiments, a cell is isolated from the embryo, the fetus, or anoffspring produced from the fetus, and a second mutation is introducedin an endogenous gene in the cell. The cell, a chromatin mass from thecell, or a nucleus from the cell is inserted into an oocyte, and theoocyte or an embryo formed from the oocyte is transferred into theuterus of a host ungulate under conditions that allow the oocyte or theembryo to develop into a fetus.

[0044] The ungulates of the invention can be used to produce antiserumor milk containing an antibody of interest. In one such aspect, theinvention features ungulate antiserum having polyclonal humanimmunoglobulins. Preferably, the antiserum is from a bovine, ovine,porcine, or caprine. In another preferred embodiment, the Igs aredirected against a desired antigen.

[0045] In yet another aspect, the invention features ungulate milkhaving polyclonal human Igs. Preferably, the milk is from a bovine,ovine, porcine, or caprine. In another preferred embodiment, the Igs aredirected against a desired antigen.

[0046] In preferred embodiments of various aspects of the invention, theheavy chain is a mu heavy chain, and the light chain is a lambda orkappa light chain. In other preferred embodiments, the nucleic acidencoding the xenogenous immunoglobulin chain or antibody is in itsunrearranged form. Preferably, the administration of an antigen ofinterest to a transgenic ungulate is followed by the rearrangement ofthe nucleic acid segments in the xenogenous immunoglobulin gene locusand the production of antibodies reactive with the antigen of interest.In other preferred embodiments, more than one class of xenogenousantibody is produced by the ungulate. In various embodiments, more thanone different xenogenous Ig or antibody is produced by the ungulate.Preferred nuclear transfer methods include inserting a cell of theinvention, a chromatin mass from the cell, or a nucleus from the cellinto an enucleated or nucleated oocyte, and transferring the oocyte oran embryo formed from the oocyte into the uterus of a host ungulateunder conditions that allow the oocyte or the embryo to develop into afetus.

[0047] In other preferred embodiments of various aspects of theinvention, the ungulate has a mutation in one or both alleles of theendogenous alpha-(1,3)-galactosyltransferase, prion, and/or J chainnucleic acid. Preferably, the mutation reduces or eliminates theexpression of the endogenous alpha-(1,3)-galactosyltransferase enzyme,galactosyl(α1,3)galactose epitope, prion protein, and/or J chain. Instill other preferred embodiments, the ungulate contains a xenogenous Jchain nucleic acid, such as a human J chain nucleic acid. Preferably,the ungulate produces human IgA or IgM molecules containing human Jchain. In various embodiments of the invention, the nucleic acid used tomutate an endogenous ungulate nucleic acid (e.g., a knockout cassettewhich includes a promoter operably linked to a nucleic acid encoding aselectable marker and operably linked to a nucleic acid havingsubstantial sequence identity to the gene to be mutated) is notcontained in a viral vector, such as an adenoviral vector or anadeno-associated viral vector. For example, the nucleic acid may becontained in a plasmid or artificial chromosome that is inserted into anungulate cell, using a standard method such as transfection orlipofection that does not involve viral infection of the cell. In yetanother embodiment, the nucleic acid used to mutate an endogenousungulate nucleic acid (e.g., a knockout cassette which includes apromoter operably linked to a nucleic acid encoding a selectable markerand operably linked to a nucleic acid having substantial sequenceidentity to the gene to be mutated) is contained in a viral vector, suchas an adenoviral vector or an adeno-associated viral vector. Accordingto this embodiment, a virus containing the viral vector is used toinfect an ungulate cell, resulting in the insertion of a portion or theentire viral vector into the ungulate cell.

[0048] Exemplary ungulates include members of the orders Perissodactylaand Artiodactyla, such as any member of the genus Bos. Other preferredungulates include sheep, big-horn sheep, goats, buffalos, antelopes,oxen, horses, donkeys, mule, deer, elk, caribou, water buffalo, camels,llama, alpaca, pigs, and elephants. Preferably, the transgenic ungulateexpresses an immunoglobulin chain or antibody from another genus, suchas an antibody from any other mammal.

[0049] As used herein, by “a nucleic acid in its pre-arranged orunrearranged form” is meant a nucleic acid that has not undergone V(D)Jrecombination. In preferred embodiments, all of the nucleic acidsegments encoding a V gene segment of an antibody light chain areseparated from all of the nucleic acid segments encoding a J genesegment by one or more nucleotides. Preferably, all of the nucleic acidsegments encoding a V gene segment of an antibody heavy chain areseparated from all of the nucleic acid segments encoding a D genesegment by one or more nucleotides, and/or all of the nucleic acidsegments encoding a D gene segment of an antibody heavy chain areseparated from all of the nucleic acid segments encoding a J genesegment by one or more nucleotides. Preferably, a nucleic acid in itsunrearranged form is substantially human. In other preferredembodiments, the nucleic acid is at least 70, 80, 90, 95, or 99%identical to the corresponding region of a naturally-occurring nucleicacid from a human.

BRIEF DESCRIPTION OF THE FIGURES

[0050]FIG. 1A contains an overview of the procedures used to produce acow that contains an Ig knockout and human artificial chromosome. Thetime line in FIG. 1A is based on an estimated 18 months to prepare theIg knockout vector and generate knockout cells, 2 months to generatefetuses from the knockout cells, 9 months to perform subsequentknockouts, 9 months of gestation for calves to be born, 12 months beforeembryos can be produced from calves, and 6 months to perform the HACtransfers.

[0051]FIG. 1B contains an overview of the methods used to produce a cowthat contains a mutation in an endogenous Ig gene and contains ΔHAC orΔΔHAC. For the time line in FIG. 1B, it is estimated that 250 coloniesare screened per week for a total of 3,000 colonies in 3 months toisolate male and female knockout cells. It is assumed that one or moreknockout colonies are produced per 1,500 colonies. Homozygous knockoutungulates may be produced by (1) introducing a second Ig mutation in anisolated knockout cell before nuclear transfer, (2) introducing a secondIg mutation in a cell obtained from a embryo, fetus (e.g., fetus at 60gestation days), or offspring produced from a first round of nucleartransfer and using the resulting homozygous cell as the donor cell in asecond round of nuclear transfer, or (3) mating hemizygous ungulates. InFIGS. 1A and 1B, “Homo” denotes homozygous; “Hemi” denotes hemizygous;“H” denotes heavy chain; “L” denotes light chain; “HAC” denotes humanartificial chromosome; “HAC 1 ” denotes either HAC; and “HAC2 ” denotesa second HAC.

[0052]FIG. 2A contains a mu (IgM heavy chain) knockout constructaccording to the invention. FIG. 2B is a restriction map ofimmunoglobulin loci from a Holstein cattle.

[0053]FIGS. 3A and 3B contain schematic illustrations of construct“pSTneoB” and “pLoxP-STneoB” that were used to produce the mu knockoutDNA construct, which is illustrated in FIG. 3C.

[0054]FIG. 3D is the polynucleotide sequence of the 1.5 kb region of thegenomic bovine mu heavy chain locus that was used as the first region ofhomology in the mu knockout construct (SEQ ID NO: 47).

[0055]FIG. 3E is the polynucleotide sequence of the 3.1 kb region of thegenomic bovine mu heavy chain locus that was used as the second regionof homology in the mu knockout construct construct (SEQ ID NO: 48). Inthis sequence, each “n” represents any nucleotide or no nucleotide. Theregion of consecutive “n” nucleotides represents an approximately 0.9 to1.0 kb region for which the polynucloetide sequence has not beendetermined.

[0056]FIG. 3F is a schematic illustration of a puromycin resistant,bovine mu heavy chain knockout construct.

[0057]FIG. 3G is the polynucleotide sequence of a bovine kappa lightchain cDNA (SEQ ID NO: 60). All or part of this sequence may be used ina kappa light chain knockout construct. Additionally, this kappa lightchain may be used to isolate a genomic kappa light chain sequence foruse in a kappa light chain knockout construct.

[0058]FIG. 4 is a schematic illustration of the construction of ΔHAC andΔΔHAC.

[0059]FIG. 5 is a picture of an agarose gel showing the presence ofgenomic DNA encoding human heavy and light chains in ΔHAC fetuses.

[0060]FIG. 6 is a picture of an agarose gel showing the expression ofhuman Cmu exons 3 and 4 in a ΔHAC fetus at 77 gestational days (fetus#5996).

[0061]FIG. 7 is a picture of an agarose gel showing the rearrangement ofendogenous bovine heavy chain in ΔHAC fetus #5996

[0062]FIG. 8 is a picture of an agarose gel showing the expression ofrearranged human heavy chain in ΔHAC fetus #5996.

[0063]FIG. 9 is a picture of an agarose gel showing the expression ofthe spliced constant region from the human heavy chain locus in ΔHACfetus #5996.

[0064]FIG. 10 is a picture of an agarose gel showing the expression ofrearranged human heavy chain in ΔHAC fetus #5996.

[0065]FIG. 11A is the polynucleotide sequence of a rearranged humanheavy chain transcript from ΔHAC fetus #5996 (SEQ ID NO: 49). FIG. 11Bis a sequence alignment of a region of this sequence (“Query”) with ahuman anti-pneumococcal antibody (“Sbjct”) (SEQ ID NOs: 50 and 51,respectively). For the query sequence from ΔHAC fetus #5996, only thosenucleotides that differ from the corresponding nucleotides of the humananti-pneumococcal antibody sequence are shown.

[0066]FIGS. 12A and 12B are two additional polynucleotide sequences (SEQID NOs: 52 and 54) and their deduced amino acid sequences (SEQ ID NOs:53 and 55, respectively) of rearranged human heavy chain transcriptsfrom ΔHAC fetus #5996.

[0067]FIG. 13 is a picture of an agarose gel demonstrating that ΔΔHACfetus #5580 contains both human heavy and light chain immunoglobulinloci.

[0068]FIG. 14 is a picture of an agarose gel demonstrating that ΔΔHACfetuses #5442A, and 5442B contain both human heavy and light chain loci.

[0069]FIG. 15 is a picture of an agarose gel showing the expression ofthe spliced mu constant region from the human heavy chain locus in ΔΔHACfetus #5542A.

[0070]FIG. 16 is a picture of an agarose gel showing the rearrangementand expression of the human heavy chain locus in ΔΔHAC fetus #5868A.

[0071]FIG. 17 is a picture of an agarose gel showing rearrangement andexpression of the human Ig lambda locus in ΔΔHAC fetuses #5442A and5442B.

[0072]FIG. 18 is a picture of an agarose gel showing rearrangement andexpression of the human Ig lambda locus in ΔΔHAC fetus #5442A.

[0073]FIG. 19 is a picture of an agarose gel showing rearrangement andexpression of the human Ig lambda locus in ΔΔHAC fetus #5868A.

[0074]FIG. 20 is a polynucleotide sequence and the corresponded deducedamino acid sequence of a rearranged human light chain transcript fromΔΔHAC fetus #5442A (SEQ ID NOs: 56 and 57, respectively).

[0075]FIG. 21 is another polynucleotide sequence and the correspondingdeduced amino acid sequence of a rearranged human light chain transcriptfrom ΔΔHAC fetus #5442A (SEQ ID NOs: 58 and 59, respectively).

[0076] FIGS. 22A-22H are graphs of a FACS analysis of expression ofhuman lambda light chain and bovine heavy chain proteins by ΔΔHACfetuses #5442A (FIGS. 22A-22D) and 5442B (FIGS. 22E-22H). Lymphocytesfrom the spleens of these fetuses were reacted with a phycoerytherinlabeled anti-human lambda antibody (FIGS. 22C and 22D), a FITC labeledanti-bovine IgM antibody (FIGS. 22D and 22H), or no antibody (FIGS. 22A,22B, (22E, and 22F) and then analyzed on a FASCalibur cell sorter. Thepercent of cells that were labeled with one of the antibodies isdisplayed beneath each histogram.

[0077]FIG. 23 is a schematic illustration of theα-(1,3)-galactosyltransferase knockout vector used to insert a puromycinresistance gene and a transcription termination sequence into theendogenous α-(1,3)-galactosyltransferase gene in bovine cells.

[0078]FIG. 24 is a schematic illustration of a BamHI-XhoI fragmentcontaining exons 2, 3, and 4 that was used as a backbone for the AAVtargeting vector. A neomycin resistance marker was used for insertionalmutagenesis of the locus by insertion into exon 4. The location of theannealing sites for the PCR primers that were used for subsequentconfirmation of appropriate targeting is indicated.

[0079]FIG. 25 is a schematic illustration of the construction of anadeno-associated viral construct designed to remove endogenous bovineIgH sequence.

[0080]FIG. 26 is a picture of an agarose gel showing the PCR analysis ofindividually transduced clones for appropriate targeting events. Thevector used in this experiment is shown in FIG. 24. PCR productsindicative of appropriate targeting are marked with asterisks.

[0081]FIG. 27 is table listing pregnancy rates for HAC carrying embryos.

DETAILED DESCRIPTION OF THE INVENTION

[0082] As discussed above, the present invention relates to theproduction of a transgenic ungulate, preferably a transgenic cow,wherein endogenous Ig expression has optionally been knocked out and anucleic acid (preferably, an artificial chromosome) has been stablyintroduced that comprises genes which are necessary for the productionof functional antibodies of another species, preferably human. Thereby,a transgenic animal may be obtained that does not produce its endogenousantibodies, but which instead produces antibodies of another species.Any non-endogenous antibodies may be produced including, withoutlimitation, human, non-human primate, dog, cat, mouse, rat, or guineapig antibodies. While the production of human monoclonal antibodies ingoats has been previously reported, this has not been effected in cows,in serum or in any ungulate that do not express its endogenousantibodies. Furthermore, the insertion of germline (unrearragned) heavyor light chain genes, the rearrangement of these genes and theexpression of diversified antibody have not been performed in atransgenic ungulate. It is unpredictable whether such ungulates wouldsurvive because it is uncertain whether human Igs will be functionallyexpressed, or expressed in sufficient amounts to provide for adequateimmune responses. Also, it is uncertain whether human chromosomes willbe stably maintained in transgenic ungulates. Still further, it isuncertain whether that ungulate (for example, bovine) B cells will beable to express or properly rearrange human or other non-endogenous Igs.

[0083] In a preferred embodiment of the present approach, xenogenousimmunoglobulin production is accomplished essentially by the combineduse of nuclear transfer, homologous recombination techniques, and theintroduction of artificial chromosomes carrying entire xenogenous Igloci. More specifically, the process preferably involves the targeteddisruption of one or both alleles of the IgM heavy chain gene, andoptionally one or both alleles of the Ig light chain gene, althoughxenogenous antibody production can also be accomplished in wild-typeanimals (i.e., animals without Ig knock outs). Gene knock outs may beeffected by sequential homologous recombination, then another matingprocedure. In a preferred embodiment, this is effected by initiallyeffecting targeted disruption of one allele of the IgM heavy chain geneof a male or female ungulate (for example, bovine) fetal fibroblast intissue culture using a suitable homologous recombination vector. The useof fetal fibroblasts is preferred over some other somatic cells as thesecells are readily propagated and genetically manipulated in tissueculture. However, the use of fetal fibroblasts is not essential to theinvention, and indeed other cell lines may be substituted therefor withequivalent results.

[0084] This process, of course, entails constructing a DNA constructhaving regions of homology to the targeted IgM heavy chain allele suchthat the construct upon integration into an IgM heavy chain allele inthe ungulate genome disrupts the expression thereof. An exemplary vectorfor carrying out such targeted disruption of an IgM allele is describedin the example which follows. In this regard, methods for constructingvectors that provide for homologous recombination at a targeted site arewell known to those skilled in the art. Moreover, in the presentinstance, the construction of a suitable vector is within the level ofskill in the art, given especially that the sequence of the bovine IgMheavy chain and Ig lambda light chain genes are known, as are thesequences of immunoglobulon genes from other ungulates (see below) Inorder to facilitate homologous recombination, the vectors used to effecthomologous recombination and inactivation of the IgM gene, respectively,comprise portions of DNA that exhibit substantial sequence identity tothe ungulate IgM heavy and Ig light chain genes. Preferably, thesesequences possessing at least 98% sequence identity, more preferably, atleast 99% sequence identity, and still more preferably will be isogenicwith the targeted gene loci to facilitate homologous recombination andtargeted deletion or inactivation.

[0085] Typically, and preferably the construct will comprise a markergene that provides for selection of desired homologous recombinants, forexample, fibroblast cells, wherein the IgM heavy chain gene and/or Iglight chain gene has been effectively disrupted. Exemplary marker genesinclude antibiotic resistance markers, drug resistance markers, andgreen fluorescent protein, among others. A preferred construct is shownin FIG. 2A and starting materials used to make this construct in FIGS.3A and 3B. Other constructs containing two regions of homology to anendogenous immunoglobulin gene, which flank a positive selection marker(e.g., an antibiotic resistance gene) that is operably linked to apromoter, may be generated using standard molecular biology techniquesand used in the methods of the present invention.

[0086] The mu knockout construct shown in FIGS. 2A and 3C was designedto remove the exons encoding the bovine immunoglobulin heavy chainconstant region, designated as “C-mu exons 1-4” and the two exonsencoding the transmembrane domain, designated “TM exons”.

[0087] To construct this vector, the region designated as “1”, anXbal-Xhol fragment from the genomic mu heavy chain bovine sequence, wassubcloned into the commercial DNA vector, pBluescript (Stratagene,LaJolla, Calif.), previously cut with the enzymes XbaI and XhoI. Oncethis fragment was cloned, there was a NotI restriction enzymerecognition sequence adjacent to the Xbal site, used to insert a NotIfragment of approximately 3.5 Kb. This fragment contains a neomycinresistance marker, described further below. If desired, other mu knockout constructs may be constructed using the genomic mu heavy chainsequence from another ungulate breed, species, or genus (e.g., the muheavy chain sequence deposited as Genbank accession number U63637 from aSwiss Bull/Holstein cross).

[0088] Once fragment “1” and the neomycin resistance marker were joinedtogether into pBluescript, there remained a Sacl site adjacent to theneomycin resistance marker. The new construct was linearized with Sacland converted to a blunt end by filling in the sticky ends left from theSacl digest, using DNA polymerase.

[0089] The fragment designated “2” was isolated as an XhoI-BstI1071fragment and converted to a blunt-ended fragment by filling in thesticky ends left from the Xhol and BstI1071 enzymes, using DNApolymerase.

[0090] Once finished, the final construct contained region 2, theneomycin resistance marker and region 1, respectively.

[0091] For transfection of bovine fibroblasts, the construct wasdigested with the restriction enzyme, Kpnl (two Kpnl sites are shown inthe diagram) and the DNA fragment was used for homologous recombination.

[0092] The neomycin resistance construct was assembled as follows. Aconstruct designated “pSTneoB” (Katoh et al., Cell Struct. Funct.12:575, 1987; Japanese Collection of Research Biologicals (JCRB) depositnumber: VE039) was designed to contain a neomycin resistance gene underthe control of an SV40 promoter and TK enhancer upstream of the codingregion. Downstream of the coding region is an SV40 terminator sequence.The neo cassette was excised from “pSTneoB” as a XhoI fragment. Afterthe ends of the fragment were converted to blunt ends using standardmolecular biology techniques, the blunt ended fragment was cloned intothe EcoRV site in the vector, pBS246 (Gibco/Life Technologies). Thissite is flanked by loxP sites. The new construct, designated“pLoxP-STNeoR”, was used to generate the mu knockout DNA construct. Thedesired fragment of this construct is flanked by loxP sites and NotIsites, which were originally present in the pBS246 cloning vector. Thedesired NotI fragment, which contains loxP-neo-loxP, was used forreplacement of the immunoglobulin mu constant region exons. The SV40promoter operably linked to the neomycin resistance gene activates thetranscription of the neomycin resistance gene, allowing cells in whichthe desired NotI fragment has replaced the mu constant region exons tobe selected based on their resulting antibiotic resistance.

[0093] After a cell line is obtained in which the IgM heavy chain allelehas been effectively disrupted, it is used as a nuclear transfer donorto produce a cloned ungulate fetus (for example, a cloned bovine fetus)and eventually a fetus or animal wherein one of the IgM heavy alleles isdisrupted. Thereafter, a second round of gene targeted disruption can beeffected using somatic cells derived therefrom, e.g., fibroblasts, inorder to produce cells in which the second IgM heavy chain allele isinactivated, using a similar vector, but containing a differentselectable marker.

[0094] Preferably, concurrent to the first targeted gene disruption, asecond ungulate (for example, bovine) somatic cell line is alsogenetically modified, which similarly may be of male or female origin.If the first cell line manipulated is male, it is preferable to modify afemale cell line; vice versa if the first cell line manipulated isfemale, it is preferable to select a male cell line. Again, preferably,the manipulated cells comprise ungulate (for example, bovine) fetalfibroblasts.

[0095] In a preferred embodiment, the female fetal fibroblast isgenetically modified so as to introduce a targeted disruption of oneallele of the Ig lambda light chain gene. This method similarly iscarried out using a vector having regions of homology to the ungulate(for example, bovine) Ig lambda light chain, and a selectable marker,which DNA construct is designed such that upon integration andhomologous recombination with the endogenous Ig light chain results indisruption (inactivation) of the targeted Ig lambda light gene.

[0096] Once a female fibroblast cell line is selected having the desiredtargeted disruption, it similarly is utilized as a donor cell fornuclear transfer or the DNA from such cell line is used as a donor fornuclear transfer.

[0097] Alternatively, this cell may be subjected to a second round ofhomologous recombination to inactivate the second Ig lambda light chainusing a similar DNA construct to that used to disrupt the first allele,but containing a different selectable marker.

[0098] As discussed in the background of the invention, methods foreffecting nuclear transfer, and particularly for the production ofcloned bovines and cloned transgenic bovines have been reported and aredescribed in U.S. Pat. No. 5,995,577 issued to Stice et al. and assignedto University of Massachusetts. Still, alternatively the nucleartransfer techniques disclosed in WO 95/16670; WO 96/07732; WO 97/0669;or WO 97/0668, (collectively, Roslin Methods) may be used. The Roslinmethods differ from the University of Massachusetts techniques in thatthey use quiescent rather than proliferating donor cells. All of thesepatents are incorporated by reference herein in their entirety. Thesenuclear transfer procedures will produce a transgenic cloned fetus whichcan be used to produce a cloned transgenic bovine offspring, forexample, an offspring which comprises a targeted disruption of at leastone allele of the Ig light chain gene and/or IgM gene. After such celllines have been created, they can be utilized to produce a male andfemale heavy and light chain hemizygous knockout (M and F Hemi H/L)fetus and offspring. Moreover, these techniques are not limited to usefor the production of transgenic bovines; the above techniques may beused for nuclear transfer of other ungulates as well.

[0099] Following nuclear transfer, production of desired animals may beaffected either by mating the ungulates or by secondary gene targetingusing the homologous targeting vector previously described.

[0100] As noted previously, a further object of the invention involvescreating male and female heavy and light chain hemizygous knockoutswherein such hemizygous knockouts are produced using the cell linesalready described. This may be affected either by mating of theoffspring produced according to the above described methods, wherein anoffspring which comprises a disrupted allele of the IgM heavy chain geneis mated with another offspring which comprises a disrupted allele ofthe Ig light chain. Alternatively, this may be affected by secondarygene targeting by manipulating a cell which is obtained from anoffspring produced according to the above-described procedures. Thiswill comprise effecting by homologous recombination targeted disruptionof an allele of the IgM heavy chain gene or allele of the Ig lightchain. After a cell line is produced which comprises a male and femaleheavy and light chain hemizygous knockout (M and F Hemi H/L) it will beused to produce a fetus or calf which comprises such a knockout. Asnoted, this is effected either by mating or secondary gene targeting.

[0101] Once the male and female heavy and light chain hemizygousknockouts are obtained, cells from these animals may be utilized tocreate homozygous knockout (Homo H/L) fetuses. Again, this is affectedeither by sequential gene targeting or mating. Essentially, if affectedby mating, this will involve mating the male heavy and light chainhemizygous knockout with a female heavy and light chain hemizygousknockout and selection of an offspring which comprises a homozygousknockout. Alternatively, the cells from the hemizygous knockoutdescribed above may be manipulated in tissue culture, so as to knock outthe other allele of the IgM or Ig light chain (lambda) gene. Secondarygene targeting may be preferred to mating as this may provide for morerapid results, especially given that the gestation period of ungulates,such as bovines, is relatively long.

[0102] Once homozygous knockouts (Homo H/L) have been obtained, they areutilized for introduction of a desired nucleic acid which contains genes(preferably, entire gene loci) for producing antibodies of a particularspecies, such as a human. Preferably, human artificial chromosomes areused, such as those disclosed in WO 97/07671 (EP 0843961) and WOOO/10383(EP 1106061). These human artificial chromosomes also are described in acorresponding issued Japanese patent JP 30300092. Both of theseapplications are incorporated by reference in their entirety herein.Also, the construction of artificial human chromosomes that contain andexpress human immunoglobulin genes is disclosed in Shen et al., Hum.Mol. Genet. 6(8):1375-1382 (1997); Kuroiwa et al., Nature Biotechnol.18(10):1086-1090 (2000); and Loupert et al., Chromosome 107(4):255-259(1998), all of which are incorporated by reference in their entiretyherein. Human artificial chromosomes may also be utilized to introducexenogenous antibody genes into wild-type animal cells; this isaccomplished using the methods described above. Introduction ofartificial chromosome into animal cells, especially fetal fibroblastcells can be performed by microcell fusion as described herein.

[0103] In an alternative to the use of human artificial chromosome,nucleic acid encoding immunoglobulin genes may be integrated into thechromosome using a YAC vector, BAC vector, or cosmid vector. Vectorscomprising xenogenous Ig genes (WO98/24893, WO96/33735, WO 97/13852,WO98/24884) can be introduced to fetal fibroblasts cells using knownmethods, such as electroporation, lipofection, fusion with a yeastspheroplast comprising a YAC vector, and the like. Further, vectorscomprising xenogenous Ig genes can be targeted to the endogenous Ig geneloci of the fetal fibroblast cells, resulting in the simultaneousintroduction of the xenogenous Ig gene and the disruption of theendogenous Ig gene.

[0104] Integration of a nucleic acid encoding a xenogenousimmunoglobulin gene may also be carried out as described in the patentsby Lonberg et al. (supra). In the “knockin” construct used for theinsertion of xenogenous immunoglobulin genes into a chromosome of a hostungulate, one or more immunoglobulin genes and an antibiotic resistancegene may be operably-linked to a promoter which is active in the celltype transfected with the construct. For example, a constitutivelyactive, inducible, or tissue specific promoter may be used to activatetranscription of the integrated antibiotic resistance gene, allowingtransfected cells to be selected based on their resulting antibioticresistance. Alternatively, a knockin construct in which the knockincassette containing the Ig gene(s) and the antibiotic resistance gene isnot operably-linked to a promoter may be used. In this case, cells inwhich the knockin cassette integrates downstream of an endogenouspromoter may be selected based on the resulting expression of theantibiotic resistance marker under the control of the endogenouspromoter. These selected cells may be used in the nuclear transferprocedures described herein to generate a transgenic ungulate containinga xenogenous immunoglobulin gene integrated into a host chromosome.

[0105] Using similar methodologies, it is possible to produce and insertartificial chromosomes containing genes for expression of Ig ofdifferent species such as dog, cat, other ungulates, non-human primatesamong other species. As discussed above, and as known in the art,immunoglobulin genes of different species are well known to exhibitsubstantial sequence homology across different species.

[0106] Once it has been determined that the inserted artificialchromosome, for example, a human artificial chromosome, has been stablyintroduced into a cell line, e.g., a bovine fetal fibroblast, it isutilized as a donor for nuclear transfer. This may be determined by PCRmethods. Similarly, animals are obtained which comprise the homozygousknockout, and further comprise a stably introduced nucleic acid, such asa human artificial chromosome. After calves have been obtained whichinclude the stably incorporated nucleic acid (for example, humanartificial chromosome), the animals are tested to determine whether theyexpress human Ig genes in response to immunization and affinitymaturation.

[0107] Modifications of the overall procedure described above may alsobe performed. For example, xenogenous Ig genes may be introduced firstand than endogenous Ig genes may be inactivated. Further, an animalretaining xenogenous Ig genes may be mated with an animal in which anendogenous Ig gene is inactivated. While the approaches to be utilizedin the invention have been described above, the techniques that areutilized are described in greater detail below. These examples areprovided to illustrate the invention, and should not be construed aslimiting. In particular, while these examples focus on transgenicbovines, the methods described may be used to produce and test anytransgenic ungulate.

Knockout Procedures to Produce Transgenic Ungulates That Express HumanIgs

[0108] As discussed above, the present invention relates to theproduction of Homo H/L fetuses or calves. The approach is summarized inFIG. 1. There are three schemes outlined therein. The first relies onsuccessive knockouts in regenerated fetal cell lines. This approach isthe technically most difficult and has the highest level of risk but asnoted above potentially yields faster results than breeding approaches.The other two schemes rely on breeding animals. In the second scheme,only single knockouts of heavy and light chain genes are required inmale and female cell lines, respectively. This scheme does not rely onregeneration of cell lines and is technically the simplest approach buttakes the longest for completion. Scheme 3 is an intermediate betweenschemes 1 and 2. In all schemes only Homo H/L fetuses are generatedbecause of potential difficulties in survival and maintenance of HomoH/L knockout calves. If necessary, passive immunotherapy can be used toincrease the survival of Homo H/L knockout calves.

[0109] Experimental Design

[0110] The present invention preferably involves the production of ahemizygous male heavy chain knockout (M Hemi H) and a hemizygous femalelight chain knockout (F Hemi L) and the production of 40 day fetusesfrom these targeted deletions. The cells from the embryos are harvested,and one allele of the light locus is targeted in the M Hemi H cells andone allele of the heavy chain locus is targeted in the F Hemi L cellsresulting in cells with hemizygous deletions of both the H and L loci(Hemi H/L). These cells are used to derive 40 day fetuses from whichfibroblasts are isolated.

[0111] The M Hemi H/L fibroblasts are targeted with the other H chainallele to create M Homo H/Hemi L, and the F Hemi H/L are targeted withthe other L chain allele to create F Homo L/Hemi H. In order to createhomozygous deletions, higher drug concentrations are used to drivehomozygous targeting. However, it is possible that this approach may notbe successful and that breeding may be necessary. An exemplary strategywhich relies on cre/lox targeting of the selection cassette allows thesame selective systems to be used for more than one targeted deletion.These fibroblasts are cloned and 40 day fetuses harvested and fibroblastcells isolated. The fetal cells from this cloning are targeted toproduce homozygous deletions of either the H or L loci resulting in MHomo H/L and F Homo H/L fetal fibroblasts. These fibroblasts are clonedand 40 day fetuses derived and fibroblasts isolated. The Homo H/L fetalfibroblasts are then used for incorporation of the HAC optionally by theuse of breeding procedures.

[0112] Library Construction

[0113] Fetal fibroblast cells are used to construct a genomic library.Although it is reported to be significant that the targeting constructbe isogenic with the cells used for cloning, it is not essential to theinvention. For example, isogenic, substantially isogenic, or nonisogenicconstructs may be used to produce a mutation in an endogenousimmunoglobulin gene. In one possible method, Holstein cattle, whichgenetically contain a high level of inbreeding compared to other cattlebreeds, are used. We have not detected any polymorphisms inimmunoglobulin genes among different animals. This suggests thatsequence homology should be high and that targeting with nonisogenicconstructs should be successful.

[0114] A library is constructed from one male cell line and one femalecell line at the same time that the “clonability” testing is beingconducted. It is envisioned that at the end of the process, a librarywill be produced and a number of different fetal cell lines will betested and one cell line chosen as the best for cloning purposes.

[0115] Genomic libraries are constructed using high molecular weight DNAisolated from the fetal fibroblast cells. DNA is size fractionated andhigh molecular weight DNA between 20-23 Kb is inserted into the lambdaphage vector LambdaZap or LambdaFix. The inventors have had excellentsuccess with Stratagene prepared libraries. Therefore, DNA is isolatedand the size selected DNA is sent to Stratagene for library preparation.To isolate clones containing bovine heavy and light chains, radiolabeledIgM cDNA and radiolabeled light chain cDNA is used. Additionally, lightchain genomic clones are isolated in case it is necessary to delete thelocus. Each fetal cell library is screened for bovine heavy and lightchain containing clones. It is anticipated that screening approximately10⁵-10⁶ plaques should lead to the isolation of clones containing eitherthe heavy chain or light chain locus. Once isolated, both loci aresubcloned into pBluescript and restriction mapped. A restriction map ofthese loci in Holsteins is provided in FIG. 2B (Knight et al. J Immunol140(10):3654-9, 1988). Additionally, a map from the clones obtained ismade and used to assemble the targeting construct.

[0116] Production of Targeting Constructs

[0117] Once the heavy and light chain genes are isolated, constructs aremade. The IgM construct is made by deleting the IgM constant regionmembrane domain. As shown by Rajewsky and colleagues in mice, deletionof the membrane domain of IgM results in a block in B cell developmentsince surface IgM is a required signal for continued B cell development(Kitamura et al., Nature 350:423-6). Thus homozygous IgM cattle lack Bcells. This should not pose a problem since in the present strategy nolive births of animals lacking functional Ig are necessary. However, ifnecessary, passive immunotherapy may be used to improve the survival ofthe animals until the last step when the human Ig loci are introduced.

[0118] An exemplary targeting construct used to effect knockout of theIgM heavy chain allele is shown below in FIG. 2A. For the heavy chain,the membrane IgM domain is replaced with a neomycin cassette flanked bylox P sites. The attached membrane domain is spliced together with theneo cassette such that the membrane domain has a TAG stop codon insertedimmediately 5′ to the lox P site ensuring that the membrane domain isinactivated. This is placed at the 5′ end of the targeting constructwith approximately 5-6 kilobases of 3′ chromosomal DNA.

[0119] If increasing drug concentrations does not allow deletion of thesecond allele of either IgM heavy or light chains, the cre/lox system(reviewed in Sauer, 1998, Methods 14:381-392) is used to delete theselectable marker. As described below, the cre/lox system allows thetargeted deletion of the selectable marker. All selectable markers areflanked with loxP sequences to facilitate deletion of these markers ifthis should be necessary.

[0120] The light chain construct contains the bovine lambda chainconstant region (e.g., the lambda light chain constant region found inGenbank accession number AF396698 or any other ungulate lambda lightchain constant region) and a puromycin resistance gene cassette flankedby lox P sites and will replace the bovine gene with a puromycincassette flanked by lox P sites. Approximately 5-6 kilobases of DNA 3′to the lambda constant region gene will be replaced 3′ to the puromycinresistance gene. The puromycin resistance gene will carry lox P sites atboth 5′ and 3′ ends to allow for deletion if necessary. Due to the highdegree of homology between ungulate antibody genes, the bovine lambdalight chain sequence in Genbank accession number AF396698 is expected tohybridize to the genomic lambda light chain sequence from a variety ofungulates and thus may be used in standard methods to isolate variousungulate lambda light chain genomic sequences. These genomic sequencesmay be used in standard methods, such as those described herein, togenerate knockout constructs to inactivate endogenous lambda lightchains in any ungulate.

[0121] A kappa light chain knockout construct may be constructedsimilarly using the bovine kappa light chain sequence in FIG. 3G or anyother ungulate kappa light chain sequence. This bovine kappa light chainmay be used as a hybridization probe to isolate genomic kappa lightchain sequences from a variety of ungulates. These genomic sequences maybe used in standard methods, such as those described herein, to generateknockout constructs to inactivate endogenous kappa light chains in anyungulate.

[0122] Additional ungulate genes may be optionally mutated orinactivated. For example, the endogenous ungulate Ig J chain gene may beknocked out to prevent the potential antigenicity of the ungulate Ig Jchain in the antibodies of the invention that are administered tohumans. For the construction of the targeting vector, the CDNA sequenceof the bovine Ig J chain region found in Genbank accession number U02301may be used. This cDNA sequence may be used as a probe to isolate thegenomic sequence of bovine Ig J chain from a BAC library such as RPC1-42(BACPAC in Oakland, Calif.) or to isolate the genomic sequence of the Jchain from any other ungulate. Additionally, the human J chain codingsequence may be introduced into the ungulates of present invention forthe functional expression of human IgA and IgM molecules. The cDNAsequence of human J chain is available from Genbank accession numbersAH002836, M12759, and M12378. This sequence may be inserted into anungulate fetal fibroblast using standard methods, such as thosedescribed herein. For example, the human J chain nucleic acid in a HAC,YAC vector, BAC vector, cosmid vector, or knockin construct may beintegrated into an endogenous ungulate chromosome or maintainedindependently of endogenous ungulate chromosomes. The resultingtransgenic ungulate cells may be used in the nuclear transfer methodsdescribed herein to generate the desired ungulates that have a mutationthat reduces or eliminates the expression of functional ungulate J chainand that contain a xenogenous nucleic acid that expresses human J chain.

[0123] Additionally, the ungulate α-(1,3)-galactosyltransferase gene maybe mutated to reduce or eliminate expression of thegalactosyl(α1,3)galactose epitope that is produced by theα-(1,3)-galactosyltransferase enzyme. If human antibodies produced bythe ungulates of the present invention are modified by this carbohydrateepitope, these glycosylated antibodies may be inactivated or eliminated,when administered as therapeutics to humans, by antibodies in therecipients that are reactive with the carbohydrate epitope. To eliminatethis possible immune response to the carbohydrate epitope, the sequenceof bovine alpha-(1,3)-galactosyltransferase gene may be used to design aknockout construct to inactive this gene in ungulates (Genbank accessionnumber J04989; Joziasse et al., J. Biol. Chem. 264(24):14290-7, 1989).This bovine sequence or the procine alpha-(1,3)-galactosyltransferasesequence disclosed in U.S. Pat. Nos. 6,153,428 and 5,821,117 may be usedto obtain the genomic alpha-(1,3)-galactosyltransferase sequence from avariety of ungulates to generate other ungulates with reduced oreliminated expression of the galactosyl(α1,3)galactose epitope.

[0124] If desired, the ungulate prion gene may be mutated or inactivatedto reduce the potential risk of an infection such as bovine spongiformencephalopathy (BSE). For the construction of the targeting vector, thegenomic DNA sequence of the bovine prion gene may be used (Genbankaccession number AJ298878). Alternatively, this genomic prion sequencemay be used to isolate the genomic prion sequence from other ungulates.The prior gene may be inactivated using standard methods, such as thosedescribed herein or those suggested for knocking out thealpha-(1,3)-galactosyltransferase gene or prion gene in sheep (Denninget al., Nature Biothech., 19: 559-562, 2001).

[0125] For targeting the second allele of each locus, it may benecessary to assemble a new targeting construct containing a differentselectable marker, if the first selectable marker remains in the cell.As described in Table 1, a variety of selection strategies are availableand may be compared and the appropriate selection system chosen.Initially, the second allele is targeted by raising the drugconcentration (for example, by doubling the drug concentration). If thatis not successful, a new targeting construct may be employed.

[0126] The additional mutations or the gene inactivation mentioned abovemay be incorporated into the ungulates of the present invention usingvarious methodologies. Once a transgenic ungulate cell line is generatedfor each desired mutation, crossbreeding may be used to incorporatethese additional mutations into the ungulates of the present invention.Alternatively, fetal fibroblast cells which have these additionalmutations can be used as the starting material for the knockout ofendogenous Ig genes and/or the introduction of xenogenous Ig genes.Also, fetal fibroblast cells having a knockout mutation in endogenous Iggenes and/or containg xenogenous Ig genes can be uses as a startingmaterial for these additional mutations or inactivations.

[0127] Targeted Deletion of Ig Loci

[0128] Targeting constructs are introduced into embryonic fibroblasts,e.g., by electroporation. The cells which incorporate the targetingvector are selected by the use of the appropriate antibiotic. Clonesthat are resistant to the drug of choice will be selected for growth.These clones are then subjected to negative selection with gancyclovir,which will select those clones which have integrated appropriately.Alternatively, clones that survive the drug selection are selected byPCR. It is estimated that it will be necessary to screen at least500-1000 clones to find an appropriately targeted clone. The inventors'estimation is based on Kitamura (Kitamura et al., Nature 350:423-6,1991) who found that when targeting the membrane domain of IgM heavychain constant region approximately 1 in 300 neo resistant clones wereproperly targeted. Thus, it is proposed to pool clones into groups of 10clones in a 96 well plate and screen pools of 10 clones for the targetedclones of choice. Once a positive is identified, single clones isolatedfrom the pooled clone will be screened. This strategy should enableidentification of the targeted clone.

[0129] Because fibroblasts move in culture it is difficult todistinguish individual clones when more than approximately ten clonesare produced per dish. Further, strategies may be developed for clonalpropagation with high efficiency transfection. Several reasonablestrategies, such as dilution cloning, may be used.

[0130] Cre/Lox Excision of the Drug Resistance Marker

[0131] As shown above, exemplary targeting constructs contain selectablemarkers flanked by loxP sites to facilitate the efficient deletion ofthe marker using the cre/lox system. Fetal fibroblasts carrying thetargeting vector are transfected via electroporation with a Crecontaining plasmid. A recently described Cre plasmid that contains aGFPcre fusion gene [Gagneten S. et al., Nucleic Acids Res 25:3326-31(1997)] maybe used. This allows the rapid selection of all clones thatcontain Cre protein. These cells are selected either by FACS sorting orby manual harvesting of green fluorescing cells via micromanipulation.Cells that are green are expected to carry actively transcribed Crerecombinase and hence delete the drug resistance marker. Cells selectedfor Cre expression are cloned and clones analyzed for the deletion ofthe drug resistance marker via PCR analysis. Those cells that aredetermined to have undergone excision are grown to small clones, splitand one aliquot is tested in selective medium to ascertain withcertainty that the drug resistance gene has been deleted. The otheraliquot is used for the next round of targeted deletion. TABLE 1Selectable markers and drugs for selection Gene Drug Neo^(r) G418¹ HphHygromycin B² Puro Puromycin³ Ecogpt Mycophenolic acid⁴ Bsr BlasticidinS⁵ HisD Histidinol⁶ DT-A Diphtheria toxin⁷

[0132] Application of Targeting Strategies to Altering ImmunoglobulinGenes of Other Ungulates

[0133] To alter immunoglobulin genes of other ungulates, targetingvectors are designed to contain three main regions. The first region ishomologous to the locus to be targeted. The second region is a drugselection marker that specifically replaces a portion of the targetedlocus. The third region, like the first region, is homologous to thetargeted locus but is not contiguous with the first region in the wildtype genome. Homologous recombination between the targeting vector andthe desired wild type locus results in deletion of locus sequencesbetween the two regions of homology represented in the targeting vectorand replacement of that sequence with a drug resistance marker. Inpreferred embodiments, the total size of the two regions of homology isapproximately 6 kilobases, and the size of the second region thatreplaces a portion of the targeted locus is approximately 2 kilobases.This targeting strategy is broadly useful for a wide range of speciesfrom prokaryotic cells to human cells. The uniqueness of each vectorused is in the locus chosen for gene targeting procedures and thesequences employed in that strategy. This approach may be used in allungulates, including, without limitation, goats (Capra hircus), sheep(Ovis aries), and the pig (Sus scrufa), as well as cattle (Bos taurus).

[0134] The use of electroporation for targeting specific genes in thecells of ungulates may also be broadly used in ungulates. The generalprocedure described herein is adaptable to the introduction of targetedmutations into the genomes of other ungulates. Modification ofelectroporation conditions (voltage and capacitance) may be employed tooptimize the number of transfectants obtained from other ungulates.

[0135] In addition, the strategy used herein to target the heavy chainlocus in cattle (i.e., removal of all coding exons and interveningsequences using a vector containing regions homologous to the regionsimmediately flanking the removed exons) may also be used equally well inother ungulates. For example, extensive sequence analysis has beenperformed on the immunoglobulin heavy chain locus of sheep (Ovis aries),and the sheep locus is highly similar to the bovine locus in bothstructure and sequence (Genbank accession numbers Z71572, Z49180 throughZ49188, M60441, M60440, AF172659 through AF172703). In addition to thelarge number of cDNA sequences reported for rearranged Ovis ariesimmunoglobulin chains, genomic sequence information has been reportedfor the heavy chain locus, including the heavy chain 5′ enhancer(Genbank accession number Z98207), the 3′ mu switch region (Z98680) andthe 5′ mu switch region (Z98681). The complete mRNA sequence for thesheep secreted form of the heavy chain has been deposited as accessionnumber X59994. This deposit contains the entire sequence of four codingexons, which are very homologous to the corresponding bovine sequence.

[0136] Information on the sheep locus was obtained from Genbank and usedto determine areas of high homology with bovine sequence for the designof primers used for PCR analysis. Because non-isogenic DNA was used totarget bovine cells, finding areas of high homology with sheep sequencewas used as an indicator that similar conservation of sequences betweenbreeds of cow was likely. Given the similarity between the sequences andstructures of the bovine and ovine immunoglobulin loci, it would beexpected that the targeting strategies used to remove bovineimmunoglobulin loci could be successfully applied to the ovine system.In addition, existing information on the pig (Sus scrofa, accessionnumber S42881) and the goat (Capra hircus, accession number AF140603),indicates that the immunoglobulin loci of both of these species are alsosufficiently similar to the bovine loci to utilize the present targetingstrategies.

Procedures for Insertion of HACs

[0137] Essentially, male and female bovine fetal fibroblast cell linescontaining human artificial chromosome sequences (#14fg., #2fg., and#22fg.) are obtained and selected and used to produce cloned calves fromthese lines.

[0138] For example, HACs derived from human chromosome #14 (“#14fg,”comprising the Ig heavy chain gene), human chromosome #2 (“#2fg,”comprising the Ig kappa chain gene) and human chromosome #22 (“#22fg,”comprising the Ig lambda chain gene) can be introduced simultaneously orsuccessively.

[0139] The transmission of these chromosome fragments is tested bymating a male #14fg. animal to female #2fg. and #22fg. animals andevaluating offspring. If transmission is successful then the two linesare mated to produce a line containing all three chromosome fragments.

[0140] Also, #14fg., #2fg., and #22fg. chromosome fragments may beinserted into Homo H/L fetal cells and used to generate cloned calves orcross transgenic HAC calves with Homo H/L calves. Alternatively, otherHACs, such as ΔHAC or ΔΔHAC, may be introduced as described in Example 2or introduced using any other chromosome transfer method.

[0141] Rationale

[0142] Germline transmission of HACs should be useful for introducingthe HACs into the Ig knockout animals and in propagating animals inproduction herds. The concern in propagation of HACs through thegermline is incomplete pairing of chromosomal material during meiosis.However, germline transmission has been successful in mice as shown byTomizuka et al. (Proc. Natl. Acad. Sci. USA, 97:722, 2000).

[0143] The strategy outlined in FIG. 1A consists of inserting #14fg.into a male line of cells and #2fg. and #22fg. each into female celllines. Calves retaining a HAC are produced and germline transmission canbe tested both through females and males. Part of the resultingoffspring (˜25%) should contain both heavy and light chain HACs. Furthercrossing should result in a line of calves containing all threechromosomal fragments. These animals are used for crossing with Homo H/Lanimals, produced from fetal cells as previously described.

[0144] Experimental Design

[0145] Cells are obtained from the original screening of cell lines.These may be Holstein or different lines than those used above. Thisallows crossing while maintaining as much genetic variation in the herdas possible. Introduction of HACs into cell lines and selection ofpositive cell lines is then effected. Selected cell lines are used fornuclear transfer and calves are produced. Starting at 12 months of agesemen and eggs are collected, fertilized, and transferred into recipientanimals. Cell samples are taken for DNA marker analysis and karyotyping.Beginning at birth, blood samples are taken and analyzed for thepresence of human Ig proteins.

[0146] As indicated above, HACs are also transferred into Homo H/L celllines using the procedures developed in the above experiments.

Testing for Human Ig Expression

[0147] The goal of the experiment is to generate male Homo H cells andcloned fetuses, to insert one or more HACs that together contain humanIgH and human IgL loci (such as HAC #14fg. and #22fg.) into Homo H cellsand generate calves, and to test expression of human Ig response toimmunization and affinity maturation. This is carried out as follows.

[0148] Experimental Design

[0149] Homo H cells are generated from Hemi H cells produced asdescribed previously. The double knockout is produced either byantibiotic selection or a second insertion. HACs are transferred intothese cells as described previously. Calves are produced by nucleartransfer. Testing calves retaining a HAC begins shortly after birth andincludes (1) evaluation for human Ig expression, (2) response toimmunization, (3) affinity maturation, and (4) transmission of the HACsto offspring.

[0150] Human Ig expression is monitored by bleeding the animals andassaying for the presence of human heavy and light chain expression byELISA, RT-PCR, or FACS analysis. Once it has been determined that theanimals produce human Ig, animals are immunized with tetanus toxoid inadjuvant. Animals are bled once a week following immunization andresponses to antigen determined via ELISA or FACS and compared topre-bleeds collected before immunization. One month after the initialimmunization, animals are boosted with an aqueous form of the antigen.One week following the boost, the animals are bled and response toantigen measured via ELISA or FACS and compared to the prebleed. TheELISA or FACS assay permits measurement of most of the titer of theresponse as well as the heavy chain isotypes produced. This data allowsa determination of an increase in antibody titer as well as theoccurrence of class switching. Estimates of average affinity are alsomeasured to determine if affinity maturation occurs during the responseto antigen.

[0151] After the transgenic bovines have been obtained as describedabove, they are utilized to produce transgenic Igs, preferably human,but potentially that of other species, e.g. dog, cat, non-human primate,other ungulates such as sheep, pig, goat, murines such as mouse, rat,guinea pig, rabbit, etc. As noted, Ig genes are known to be conservedacross species.

Transgenic Antisera and Milk Containing Xenogenous Antibodies

[0152] The bovine (or other ungulate) yields transgenic antiseradirected to whatever antigen(s) it is endogenously exposed, or toexogenously administered antigen(s). For example, antigens may beadministered to the ungulate to produce desired antibodies reactive withthe antigens, including antigens such as pathogens (for example,bacteria, viruses, protozoans, yeast, or fungi), tumor antigens,receptors, enzymes, cytokines, etc. Exemplary pathogens for antibodyproduction include, without limitation, hepatitis virus (for example,hepatitis C), immunodeficiency virus (for example, HIV), herpes virus,parvovirus, enterovirus, ebola virus, rabies virus, measles virus,vaccinia virus, Streptococcus (for example, Streptococcus pneumoniae),Haemaphilus (for example, Haemophilus influenza), Neisseria (forexample, Neisseria meningitis), Coryunebacterium diptheriae, Haemophilus(for example, Haemophilus pertussis), Clostridium (for example,Clostridium botulinium), Staphlococcus, Pseudomonas (for example,Pseudomonas aeruginosa), and respiratory syncytial virus (RSV).

[0153] One or more pathogens may be administered to a transgenicungulate to generate hyperimmune serum useful for the prevention,stabilization, or treatment of a specific disease. For example,pathogens associated with respiratory infection in children may beadministered to a transgenic ungulate to generate antiserum reactivewith these pathogens (e.g., Streptococcus pneumoniae, Haemophilusinfluenza, and/or Neissaria meningitis). These pathogens may optionallybe treated to reduce their toxicity (e.g., by exposure to heat orchemicals such as formaldehyde) prior to administration to the ungulate.

[0154] For the generation of broad spectrum Ig, a variety of pathogens(e.g., multiple bacterial and/or viral pathogens) may be administered toa transgenic ungulate. This hyperimmune serum may be used to prevent,stabilize, or treat infection in mammals (e.g., humans) and isparticularly useful for treating mammals with genetic or acquiredimmunodeficiencies.

[0155] In addition, antibodies produced by the methods of the inventionmay be used to suppress the immune system, for example, to treatneuropathies, as well as to eliminate particular human cells andmodulate specific molecules. For example, anti-idiotypic antibodies(i.e., antibodies which inhibit other antibodies) and antibodiesreactive with T cells, B cells, or cytokines may be useful for thetreatment of autoimmune disease or neuropathy (e.g., neuropathy due toinflammation). These antibodies may be obtained from transgenicungulates that have not been administered an antigen, or they may beobtained from transgenic ungulates that have been administered anantigen such as a B cell, T cell, or cytokine (e.g., TNFα).

[0156] Transgenic antisera generated from transgenic ungulates that havenot been administered an antigen may be used to manufacturepharmaceuticals comprising human polyclonal antibodies, preferably humanIgG molecules. These human antibodies may be used in place of antibodiesisolated from humans as Intraveneous Immunoglobulin (IVIG) therapeutics.

[0157] Transgenic antiserum may optionally be enriched for antibodiesreactive against one or more antigens of interest. For example, theantiserum may be purified using standard techniques such as thosedescribed by Ausubel et al. (Current Protocols in Molecular Biology,volume 2, p. 11.13.1-11.13.3, John Wiley & Sons, 1995). Preferredmethods of purification include precipitation using antigen or antibodycoated beads, column chromatography such as affinity chromatography,magnetic bead affinity purification, and panning with a plate-boundantigen. Additionally, the transgenic antiserum may be contacted withone or more antigens of interest, and the antibodies that bind anantigen may be separated from unbound antibodies based on the increasedsize of the antibody/antigen complex. Protein A and/or protein G mayalso be used to purify IgG molecules. If the expression of endogenousantibodies is not eliminated, protein A and/or an antibody against humanIg light chain lambda (Pharmingen) may be used to separate desired humanantibodies from endogenous ungulate antibodies or ungulate/humanchimeric antibodies. Protein A has higher affinity for human Ig heavychain than for bovine Ig heavy chain and may be used to separate desiredIg molecules containing two human heavy chains from other antibodiescontaining one or two ungulate heavy chains. An antibody against humanIg light chain lambda may be used to separate desired Ig moleculeshaving two human Ig lambda chains from those having one or two ungulateIg light chains. Additionally or alternatively, one or more antibodiesthat are specific for ungulate Ig heavy or light chains may be used in anegative selection step to remove Ig molecules containing one or twoungulate heavy and/or light chains.

[0158] The resultant antisera may itself be used for passiveimmunization against an antigen. Alternatively, the antisera hasdiagnostic, prophylactic, or purification use, e.g. for attainingpurification of antigens.

[0159] Alternatively, after antisera administration, B cells may beisolated from the transgenic bovine and used for hybridoma preparation.For example, standard techniques may be used to fuse a B cell from atransgenic ungulate with a myeloma to produce a hybridoma secreting amonoclonal antibody of interest (Mocikat, J. Immunol. Methods225:185-189, 1999; Jonak et al., Hum. Antibodies Hybridomas 3:177-185,1992; Srikumaran et al., Science 220:522, 1983). Preferred hybridomasinclude those generated from the fusion of a B-cell with a myeloma froma mammal of the same genus or species as the transgenic ungulate. Otherpreferred myelomas are from a Balb/C mouse or a human. In this instance,hybridomas are provided that make xenogenous monoclonal antibodiesagainst a particular antigen. For example, this technology may be usedto produce human, cat, dog, etc. (dependent upon the specific artificialchromosome) monoclonal antibodies that are specific to pathogens.Methods for selecting hybridomas that produce antibodies havingdesirable properties, i.e., enhanced binding affinity, avidity, are wellknown.

[0160] Alternatively, a B cell from a transgenic ungulate may begenetically modified to express an oncogene, such as ras, myc, abl,bcl2, or neu, or infected with a transforming DNA or RNA virus, such asEpstein Barr virus or SV40 virus (Kumar et al., Immunol. Lett.65:153-159, 1999; Knight et al., Proc. Nat. Acad. Sci. USA 85:3130-3134,1988; Shammah et al., J. Immunol. Methods 160-19-25, 1993; Gustafssonand Hinkula, Hum. Antibodies Hybridomas 5:98-104, 1994; Kataoka et al.,Differentiation 62:201-211, 1997; Chatelut et al., Scand. J. Immunol.48:659-666, 1998). The resulting immortalized B cells may also be usedto produce a theoretically unlimited amount of antibody. Because Ig isalso secreted into the milk of ungulates, ungulate milk may also be usedas a source of xenogenous antibodies.

[0161] While the invention has been described adequately supra, thefollowing examples are additionally provided as further exemplificationof the invention.

EXAMPLE 1 Bovine IgM Knock Out

[0162] The following procedures were used to generate bovine fibroblastcell lines in which one allele of the immunoglobulin heavy chain (mu)locus is disrupted by homologous recombination. A DNA construct foreffecting an IgM knockout was generated by the removal of exons 1-4 ofthe Mu locus (corresponds to IgM heavy chain gene) which were replacedwith a copy of a neomycin resistance gene. Using this construct,neomycin resistant cell lines have been obtained which were successfullyused in nuclear transfer procedures, and blastocysts from these celllines have been implanted into recipient cows. Additionally, some ofthese blastocysts were tested to confirm that targeted insertionoccurred appropriately in the mu locus using PCR procedures. Blastocystsresulting from nuclear transfer procedures from several of the celllines obtained indicated that heterozygous IgM-KO fetuses were ingestation. Additionally, both male and female cell lines that comprise asingle IgM heavy chain (mu) knockout have been produced. It isanticipated that mating of animals cloned from these cell lines willgive rise to progeny wherein both copies of mu are inactivated. Theseprocedures are discussed in greater detail below.

[0163] DNA Construct

[0164] The DNA used in all transfections described in this document wasgenerated as follows. The four main exons (excluding the transmembranedomain exons), CH1-4, are flanked by an XhoI restriction site at thedownstream (CH4) end and an XbaI site at the upstream (CH1) end. Theconstruct used for the transfection procedure consisted of 1.5 kb ofgenomic sequence downstream of the XhoI site and 3.1 Kb of genomicsequence upstream of the XbaI site (FIGS. 3D and 3E). These sequenceswere isolated as described herein from a Holstein cow from a dairy herdin Massachusetts. A neomycin resistance marker was inserted betweenthese two fragments on a 3.5 Kb fragment, replacing 2.4 Kb of DNA,originally containing CH1-4, from the originating genomic sequence. Thebackbone of the vector was pBluescriptIl SK+ (Stratagene) and the insertof 8.1 Kb was purified and used for transfection of bovine fetalfibroblasts. This construct is shown in FIGS. 3A-3C. Other mu knockoutconstructs containing other homologous regions and/or containing anotherantibiotic resistance gene may also be constructed using standardmethods and used to mutate an endogenous mu heavy chain gene.

[0165] Transfection /Knockout Procedures

[0166] Transfection of fetal bovine was performed using a commercialreagent, Superfect Transfection Reagent (Qiagen, Valencia, Calif., USA),Catalog Number 301305.

[0167] Bovine fibroblasts were generated from disease-tested maleCharlais cattle at Hematech's Kansas facility and sent to Hematech'sWorcester Molecular Biology Labs for use in all experiments described.Any other ungulate breed, genus, or species may be used as the source ofdonor cells (e.g., somatic cells such as fetal fibroblasts). The donorcells are genetically modified to contain a mutation that reduces oreliminates the expression of functional, endogenous Ig.

[0168] The medium used for culture of bovine fetal fibroblasts consistedof the following components: 500 ml Alpha MEM (Bio-Whittaker # 12-169F);50 ml fetal calf serum (Hy-Clone #A-1111-D); 2 ml antibiotic/antimyotic(Gibco/BRL #15245-012); 1.4 ml 2-mercaptoethanol (Gibco/BRL #21985-023);5.0 ml L-Glutamine (Sigma Chemical #G-3126); and 0.5 ml tyrosinetartrate (Sigma Chemical #T-6134).

[0169] On the day prior to transfection procedures, cells were seeded in60 mm tissue culture dishes with a targeted confluency of 40-80% asdetermined by microscopic examination.

[0170] On the day of transfection, 5 μg of DNA, brought to a totalvolume of 150 μl in serum-free, antibiotic-free medium, was mixed with20 μl of Superfect transfection reagent and allowed to sit at roomtemperature for 5-10 minutes for DNA-Superfect complex formation. Whilethe complex formation was taking place, medium was removed from the 60mm tissue culture dish containing bovine fibroblasts to be transfected,and cells were rinsed once with 4 ml of phosphate-buffered saline. Onemilliliter of growth medium was added to the 170 μl DNA/Superfectmixture and immediately transferred to the cells in the 60 mm dish.Cells were incubated at 38.5° C., 50% carbon dioxide for 2.5 hours.After incubation of cells with the DNA/Superfect complexes, medium wasaspirated off and cells were washed four times with 4 ml PBS. Five ml ofcomplete medium were added and cultures were incubated overnight at38.5° C., 5% CO₂. Cells were then washed once with PBS and incubatedwith one ml of 0.3% trypsin in PBS at 37° C. until cells were detachedfrom the plate, as determined by microscopic observation. Cells fromeach 60 mm dish were split into 24 wells of a 24 well tissue cultureplate (41.7 ul/well). One milliliter of tissue culture medium was addedto each well and plates were allowed to incubate for 24 hours at 38.5°C. and 5% CO₂ for 24 hours.

[0171] During all transfection procedures, sham transfections wereperformed using a Superfect/PBS mixture containing no DNA, as none ofthose cells would be expected to contain the neomycin resistance geneand all cells would be expected to die after addition of G418 to thetissue culture medium. This served as a negative control for positiveselection of cells that received DNA.

[0172] After the 24 hour incubation, one more milliliter of tissueculture medium containing 400 μg G418 was added to each well, bringingthe final G418 concentration to 200 μg/ml. Cells were placed back intothe incubator for 7 days of G418 selection. During that period, bothtransfected and sham transfection plates were monitored for cell deathand over 7 days, the vast majority of wells from the sham transfectionscontained few to no live cells while plates containing cells thatreceived the DNA showed excellent cell growth.

[0173] After the 7 day selection period, the cells from wells at 90-100%confluency were detached using 0.2 ml 0.3% trypsin in PBS and weretransferred to 35 mm tissue culture plates for expansion and incubateduntil they became at least 50% confluent, at which point, cells weretrypsinized with 0.6 ml 0.3% trypsin in PBS. From each 35 mm tissueculture plate, 0.3 ml of the 0.6 ml cell suspension was transferred to a12.5 cm² tissue culture flask for further expansion. The remaining 0.3ml was reseeded in 35 mm dishes and incubated until they attained aminimal confluency of approximately 50%, at which point cells from thoseplates were processed for extraction of DNA for PCR analysis. Flasksfrom each line were retained in the incubator until they had undergonethese analyses and were either terminated if they did not contain thedesired DNA integration or kept for future nuclear transfer andcryopreservation.

[0174] Screening for Targeted Integrations

[0175] As described above the DNA source for screening of transfectantscontaining the DNA construct was a 35 mm tissue culture dish containinga passage of cells to be analyzed. DNA was prepared as follows and isadapted from a procedure published by Laird et al. (Laird et al.,“Simplified mammalian DNA isolation procedure”, Nucleic Acids Research,19:4293). Briefly, DNA was prepared as follows. A cell lysis buffer wasprepared with the following components: 100 mM Tris-HCl buffer, pH 8.5;5 mM EDTA, pH 8.0; 0.2% sodium dodecyl sulfate; 200 mM NaCl; and 100ug/ml Proteinase K.

[0176] Medium was aspirated from each 35 mm tissue culture dish andreplaced with 0.6 ml of the above buffer. Dishes were placed back intothe incubator for three hours, during which time cell lysis and proteindigestion were allowed to occur. Following this incubation, the lysatewas transferred to a 1.5 ml microfuge tube and 0.6 ml of isopropanol wasadded to precipitate the DNA. Tubes were shaken thoroughly by inversionand allowed to sit at room temperature for 3 hours, after which the DNAprecipitates were spun down in a microcentrifuge at 13,000 rpm for tenminutes. The supernatant from each tube was discarded and the pelletswere rinsed with 70% ethanol once. The 70% ethanol was aspirated off andthe DNA pellets were allowed to air-dry. Once dry, each pellet wasresuspended in 30-50 ul of Tris (10 mM)-EDTA (1 mM) buffer, pH 7.4 andallowed to hydrate and solubilize overnight. 5-7 microliters of each DNAsolution was used for each polymerase chain reaction (PCR) procedure.

[0177] Two separate PCR procedures were used to analyze transfectants.The first procedure used two primers that were expected to anneal tosites that are both located within the DNA used for transfection. Thefirst primer sequence is homologous to the neomycin resistance cassetteof the DNA construct and the second is located approximately 0.5 Kbaway, resulting in a short PCR product of 0.5 Kb. In particular, primersNeol (5′-CTT GAA GAC GAA AGG GCC TCG TGA TAC GCC-3′, SEQ ID NO: 42) andIN2521 (5′-CTG AGA CTT CCT TTC ACC CTC CAG GCA CCG-3′, SEQ ID NO: 43)were used. A Qiagen PCR kit was used for this PCR reaction. The PCRreaction mixture contained 1 pmole of each primer, 5 ul of 10× reactionbuffer, 10 ul of Q solution, 5 ul of DNA, and 1 ul of dNTP solution. Thereaction mixture was brought to a total volume of 50 ul with H₂O. ThisPCR amplification was performed using an initial denaturing incubationat 94° C. for two minutes. Then, 30 cycles of denaturation, annealing,and amplification were performed by incubation at 94° C. for 45 seconds,60° C. for 45 seconds, and 72° C. for two minutes. Then, the reactionmixture was incubated at 72° C. for five minutes and at 4° C. until themixture was removed from the PCR machine. Alternatively, any otherprimers that are homologous to the region of the knockout construct thatintegrates into the genome of the cells may be used in a standard PCRreaction under appropriate reaction conditions to verify that cellssurviving G418 selection were resistant as a result of integration ofthe DNA construct.

[0178] Because only a small percentage of transfectants would beexpected to contain a DNA integration in the desired location (the Mulocus), another pair of primers was used to determine not only that theDNA introduced was present in the genome of the transfectants but alsothat it was integrated in the desired location. The PCR procedure usedto detect appropriate integration was performed using one primer locatedwithin the neomycin resistance cassette of the DNA construct and oneprimer that would be expected to anneal over 1.8 Kb away, but only ifthe DNA had integrated at the appropriate site of the IgM locus (sincethe homologous region was outside the region included in the DNAconstruct used for transfection). The primer was designed to anneal tothe DNA sequence immediately adjacent to those sequences represented inthe DNA construct if it were to integrate in the desired location (DNAsequence of the locus, both within the region present in the DNAconstruct and adjacent to them in the genome was previously determined).In particular, primers Neol and OUT3570 (5′-CGA TGA ATG CCC CAT TTC ACCCAA GTC TGT C-3′, SEQ ID NO: 44) were used for this analysis. This PCRreaction was performed using a Qiagen PCR kit as described above for thefirst PCR reaction to confirm the integration of the targeting constructinto the cells. Alternatively, this PCR analysis may be performed usingany appropriate reaction conditions with any other primer that ishomologous to a region of the knockout construct that integrates intothe genome of the cells and any other primer that is homologous to aregion in the genome of the cells that is upstream or downstream of thesite of integration.

[0179] Using these methods, 135 independent 35 mm plates were screenedfor targeted integration of the DNA construct into the appropriatelocus. Of those, DNA from eight plates was determined to contain anappropriately targeted DNA construct and of those, three were selectedfor use in nuclear transfer procedures. Those cells lines weredesignated as “8-1C”, “5-3C” and “10-1C”. Leftover blastocysts not usedfor transfer into recipient cows were used to extract DNA which wassubjected to additional PCR analysis. This analysis was effective usinga nested PCR procedure using primers that were also used for initialscreening of transfected lines.

[0180] As noted above, three cell lines were generated using the genetargeting construct designed to remove exons 1-4 of the mu locus. Theselines all tested positive for targeted insertions using a PCR based testand were used for nuclear transfers. Leftover blastocysts resulting fromthose nuclear transfers were screened by PCR testing the appropriatelytargeted construct. The following frequencies of positive blastocystswere obtained: Cell Line 8-1C: 6/8 Cell Line 10-1C: 2/16 Cell Line 5-3C:0/16

[0181] Although at forty days of gestation, 11 total pregnancies weredetected by ultrasound, by day 60, 7 fetuses had died. The remaining 4fetuses were processed to regenerate new fetal fibroblasts and remainingorgans were used to produce small tissue samples for PCR analysis. Theresults of the analyses are below: Line 8-1C: two fetuses, one fetuspositive for targeted insertion by PCR Line 10-1C: one fetus, positivefor targeted insertion by PCR Line 5-3C: one fetus, negative fortargeted insertion by PCR

[0182] Surprisingly, although the frequency of 10-1 C blastocyststesting positive for targeted insertion was only 2/16, the one viable60-day fetus obtained from that cell line was positive as determined byPCR. A positive fetus from 8-1C was also obtained. Southern blotanalysis of DNA of all tissue samples is being effected to verify thatthe construct not only targeted correctly at one end (which isdetermined by PCR of the shorter region of homology present in theoriginal construct) but also at the other end. Based on results to date,it is believed that two heavy chain knockout fetuses from twoindependent integration events have been produced. Also, since thesefetuses were derived from two different lines, at least one is likely tohave integrated construct correctly at both ends. Once the Southern blotanalyses have confirmed appropriate targeting of both ends of targetingconstruct, further nuclear transfers will be performed to generateadditional fetuses which will be carried to term.

[0183] Nuclear Transfer and Embryo Transfer

[0184] Nuclear transfers were performed with the K/O cell line (8-1-C(18)) and eight embryos were produced. A total of six embryos from thisbatch were transferred to three disease free recipients at Trans OvaGenetics (“TOG”; Iowa).

[0185] Frozen embryos have been transferred to ten disease freerecipients to obtain disease free female fibroblast cell lines. Fetalrecoveries are scheduled after confirming the pregnancies at 35-40 days.

[0186] Pregnancy Diagnosis and Fetal Recovery

[0187] Pregnancy status of the eighteen recipients transferred withcloned embryos from knockout fetal cells was checked by ultrasonography.The results are summarized below. TABLE 2 Pregnancy at 40 days using muheavy chain knockout donor cells Clone ID No of recips transferredPregnancy at 40 days (%) 8-1-0C 5 4 (80) 10-1-C 6 4 (67) 5-3-C 5 3 (60)Total 16 11 (69) 

[0188] Pregnancy Diagnosis

[0189] Pregnancy status of the three recipients to whom cloned embryoswere transferred from knockout cells (8-1C) was checked; one was openand the other two required reconfirmation after one month.

[0190] Fetal Recoveries and Establishment of Cell Lines

[0191] Eleven pregnancies with the K/O embryos at 40 days were obtained.Four live fetuses were removed out of these at 60 days. Cell lines wereestablished from all four and cryopreserved for future use. Also wecollected and snap froze tissue samples from the fetuses and sent themto Hematech molecular biology laboratory for PCR/Southern blot analysis.

[0192] All four of the cell lines were male. In order to secure a femalecell line, cell lines were established and cryopreserved for futureestablishment of K/O cells from the fetuses (six) collected at 55 daysof gestation from the pregnancies established at Trans Ova Genetics withdisease free recipients. Recently, the existence of a female cell linecontaining a mu knockout was confirmed. This female cell line may beused to produce cloned animals which may be mated with animals generatedfrom the male cell lines, and progeny screened for those that containthe double mu knockout.

EXAMPLE 2 Introduction of HAC

[0193] Additional experiments were carried out to demonstrate thatimmunoglobulin heavy chain (mu) and lambda light chain may be producedby a bovine host, either alone or in combination. In addition, theseexperiments demonstrated that the immunoglobulin chains were rearrangedand that polyclonal sera was obtained. In these procedures,immunoglobulin-expressing genes were introduced into bovine fibroblastsusing human artificial chromosomes. The fibroblasts were then utilizedfor nuclear transfer, and fetuses were obtained and analyzed forantibody production. These procedures and results are described in moredetail below.

[0194] HAC Constructs

[0195] The human artificial chromosomes (HACs) were constructed using apreviously described chromosome-cloning system (Kuroiwa et al., NatureBiotech. 18: 1086-1090, 2000). Briefly, for the construction of ΔHAC,the previously reported human chromosome 22 fragment (hChr22) containinga loxP sequence integrated at the HCF2 locus was truncated at theAP000344 locus by telomere-directed chromosomal truncation (Kuroiwa etal., Nucleic Acid Res., 26: 3447-3448, 1998). Next, cell hybrids wereformed by fusing the DT40 cell clone containing the above hChr22fragment (hCF22) truncated at the AP000344 locus with a DT40 cell clone(denoted “R clone”) containing the stable and germline-transmittablehuman minichromosome SC20 vector. The SC20 vector was generated byinserting a loxP sequence at the RNR2 locus of the S20 fragment. TheSC20 fragment is a naturally-occurring fragment derived from humanchromosome 14 that includes the entire region of the human Ig heavychain gene (Tomizuka et al., Proc. Natl. Acad. Sci. USA 97:722, 2000).The resulting DT40 cell hybrids contained both hChr fragments. The DT40hybrids were transfected with a Cre recombinase-expression vector toinduce Cre/loxP-mediated chromosomal translocation between hCF22 and theSC20 vector. The stable transfectants were analyzed using nested PCR toconfirm the cloning of the 2.5 megabase hChr22 region, defined by theHCF2 and AP000344 loci, into the loxP-cloning site in the SC20 vector.The PCR-positive cells which were expected to contain ΔHAC were thenisolated by FACS sorting based on the fluorescence of the encoded greenfluorescent protein. Fluorescent in situ hybridization (FISH) analysisof the sorted cells was also used to confirm the presence of ΔHAC, whichcontains the 2.5 megabase hChr22 insert.

[0196] Similarly, ΔΔHAC was also constructed using thischromosome-cloning system. The hChr22 fragment was truncated at theAP000344 locus, and then the loxP sequence was integrated into theAP000553 locus by homologous recombination in DT40 cells. The resultingcells were then fused with the R clone containing the SC20minichromosome vector. The cell hybrids were transfection with aCre-expression vector to allow Cre/loxP-mediated chromosomaltranslocation. The generation of ΔΔHAC, which contains the 1.5 megabasehChr22 insert, defined by the AP000553 and AP000344 loci, was confirmedby PCR and FISH analyses.

[0197] The functionality of ΔHAC and ΔΔHAC in vivo was assessed by thegeneration of chimeric mice containing these HACs. These HACs wereindividually introduced into mouse embryonic stem (ES) cells, which werethen used for the generation of chimeric mice using standard procedures(Japanese patent number 2001-142371; filed May 11, 2000). The resultingmice had a high degree of chimerism (85-100% of coat color),demonstrating a high level of pluripotency of the ES cells containingthese HACs and the mitotic stability of these HACs in vivo. Furthermore,ΔHAC was transmitted through the germline of the ΔHAC chimeric mouse tothe next offspring, demonstrating the meiotic stability of this HAC.

[0198] Chicken DT40 cells retaining these HACs have been deposited underthe Budapest treaty on May 9, 2001 in the International Patent OrganismDepository, National Institute of Advanced Industrial Science andTechnology, Tsukuba Central 6, 1-1, Higashi 1-Chome Tsukuba-shi,Ibaraki-ken, 305-8566 Japan. The depository numbers are as follows: ΔHAC(FERM BP-7582), ΔΔHAC (FERM BP-7581), and SC20 fragment (FERM BP-7583).Chicken DT40 cells retaining these HACs have also been deposited in theFood Industry Research and Development Institute (FIRDI) in Taiwan. Thedepository numbers and dates are as follows: ΔHAC (CCRC 960144; Nov. 9,2001), ΔΔHAC (CCRC 960145; Nov. 9, 2001), and SC20 fragment (the cellline was deposited under the name SC20 (D); CCRC 960099; Aug. 18, 1999).

[0199] The 2.5 megabase (Mb) hChr22 insert in ΔHAC is composed of thefollowing BAC contigs, which are listed by Genbank accession number:AC002470, AC002472, AP000550, AP000551, AP000552, AP000556, AP000557,AP000558, AP000553, AP000554, AP000555, D86995, D87019, D87012, D88268,D86993, D87004, D87022, D88271, D88269, D87000, D86996, D86989, D88270,D87003, D87018, D87016, D86999, D87010, D87009, D87011, D87013, D87014,D86991, D87002, D87006, D86994, D87007, D87015, D86998, D87021, D87024,D87020, D87023, D87017, AP000360, AP00361, AP000362, AC000029, AC000102,U07000, AP000343, and AP000344. The 1.5 Mb hChr22 insert in ΔΔHAC iscomposed of the following BAC contigs: AP000553, AP000554, AP000555,D86995, D87019, D87012, D88268, D86993, D87004, D87022, D88271, D88269,D87000, D86996, D86989, D88270, D87003, D87018, D87016, D86999, D87010,D87009, D87011, D87013, D87014, D86991, D87002, D87006, D86994, D87007,D87015, D86998, D87021, D87024, D87020, D87023, D87017, AP000360,AP00361, AP000362, AC000029, AC000102, U07000, AP000343, and AP000344(Dunham et al, Nature 402:489-499, 1999).

[0200] Generation of Bovine Fetal Fibroblasts To generate bovine fetalfibroblasts, day 45 to 60 fetuses were collected from disease-testedHolstein or Jersey cows housed at Trans Ova (Iowa), in which thepedigree of the male and female parents were documented for threeconsecutive generations. The collected fetuses were shipped on wet iceto Hematech's Worcester Molecular Biology Division for the generation ofprimary fetal fibroblasts. Following arrival, the fetus(es) weretransferred to a non-tissue culture grade, 100 mm plastic petri dish ina tissue culture hood. Using sterile forceps and scissors, theextraembryonic membrane and umbilical cord were removed from the fetus.After transferring the fetus to a new plastic petri dish, the head,limbs and internal organs were removed. The eviscerated fetus wastransferred to a third petri dish containing approximately 10 ml offetus rinse solution composed of: 125 ml 1× Dulbecco's-PBS (D-PBS) withCa²⁺ and Mg²⁺ (Gibco-BRL, cat#. 14040); 0.5 ml Tylosine Tartrate (8mg/ml, Sigma, cat#. T-3397); 2 ml Penicillin-Streptomycin (Sigma, cat#.P-3539); and 1 ml of Fungizone (Gibco-BRL, cat#. 15295-017) (mixed andfiltered through a 0.2 μm nylon filter unit [Nalgene, cat#. 150-0020).

[0201] The fetus was washed an additional three times with the fetusrinse solution to remove traces of blood, transferred to a 50 ml conicaltissue culture tube, and finely minced into small pieces with a sterilescalpel. The tissue pieces were washed once with 1× D-PBS without Ca²⁺and Mg²⁺ (Gibco-BRL, cat#. 14190). After the tissue pieces settled tothe bottom of the tube, the supernatant was removed and replaced withapproximately 30 ml of cell dissociation buffer (Gibco-BRL, cat#.13151-014). The tube was inverted several times to allow mixing andincubated at 38.5° C./5% C0₂ for 20 minutes in a tissue cultureincubator. Following settling of the tissue to the bottom of the tube,the supernatant is removed and replaced with an equivalent volume offresh cell dissociation buffer. The tissue and cell dissociation buffermixture was transferred to a sterile, 75 ml glass trypsinizing flask(Wheaton Science Products, cat#. 355393) containing a 24 mm,round-ended, spin bar. The flask was transferred to a 38.5° C./5% CO₂tissue culture incubator, positioned on a magnetic stir plate, andstirred at a sufficient speed to allow efficient mixing forapproximately 20 minutes. The flask was transferred to a tissue culturehood; the tissue pieces allowed to settle, followed by removal of thesupernatant and harvesting of the dissociated cells by centrifugation at1,200 rpm for five minutes. The cell pellet was re-suspended in a smallvolume of complete fibroblast culture media composed of: 440 ml alphaMEM (BioWhittaker, cat#. 12-169F); 50 ml irradiated fetal bovine serum;5 ml GLUTAMAX-I supplement (Gibco-BRL, cat#. 25050-061); 5 mlPenicillin-Streptomycin (Sigma, cat#. P-3539); 1.4 ml 2-mercaptoethanol(Gibco-BRL, cat#. 21985-023) (all components except the fetal bovineserum were mixed were filtered through 0.2 μm nylon filter unit[Nalgene, cat#. 151-4020]), and stored on ice. The dissociation processwas repeated three additional times with an additional 30 ml of celldissociation solution during each step. Cells were pooled; washed incomplete fibroblast media; passed sequentially through 23 and 26 gaugeneedles, and finally through a 70 μm cell strainer (B-D Falcon, cat#.352350) to generate a single cell suspension. Cell density and viabilitywere determined by counting in a hemacytometer in the presence of trypanblue (0.4% solution, Sigma, cat#. T-8154).

[0202] Primary fibroblasts were expanded at 38.5° C./5% CO₂ in completefibroblast media at a cell density of 1×10⁶ viable cells per T75 cm²tissue culture flask. After 3 days of culture or before the cellsreached confluency, the fibroblasts were harvested by rinsing the flaskonce with 1× D-PBS (without Ca²⁺ and Mg²⁺) and incubating with 10 ml ofcell dissociation buffer for 5 to 10 minutes at room temperature.Detachment of cells was visually monitored using an inverted microscope.At this step, care was taken to ensure that cell clumps weredisaggregated by pipeting up-and-down. After washing and quantitation,the dissociated fibroblasts were ready for use in gene targetingexperiments. These cells could also be cryopreserved for long-termstorage.

[0203] Introduction of HACs into Bovine Fetal Fibroblasts

[0204] ΔHAC and ΔΔHAC were transferred from the DT40 cell hybrids toChinese hamster ovary (CHO) cells using microcell-mediated chromosometransfer (MMCT) (Kuroiwa et al. Nature Biotech. 18: 1086-1090, 2000).The CHO clone containing ΔHAC (“D15 clone”) was cultured in F12 (Gibco)medium supplemented with 10% FBS (Gibco), 1 mg/ml of G418, and 0.2 mg/mlof hygromycin B at 37° C. and 5% CO₂. The D15 clone was expanded intotwelve T25 flasks. When the confluency reached 80-90%, colcemid (Sigma)was added to the medium at a final concentration of 0.1 μg/ml. Afterthree days, the medium was exchanged with DMEM (Gibco) supplemented with10 μg/ml of cytochalacin B (Sigma). The flasks were centrifuged for 60minutes at 8,000 rpm to collect microcells. The microcells were purifiedthrough 8, 5, and 3-μm filters (Costar) and then resuspended in DMEMmedium. The microcells were used for fusion with bovine fibroblasts asdescribed below.

[0205] Bovine fetal fibroblasts were cultured in α-MEM (Gibco) mediumsupplemented with 10% FBS (Gibco) at 37° C. and 5% CO₂. The fibroblastswere expanded in a T175 flask. When the confluency reached 70-80%, thecells were detached from the flask with 0.05% trypsin. The fibroblastcells were washed twice with DMEM medium and then overlayed on themicrocell suspension. After the microcell-fibroblast suspension wascentrifuged for five minutes at 1,500 rpm, PEG1500 (Roche) was added tothe pellet according to the manufacturer's protocol to enable fusion ofthe microcells with the bovine fibroblasts. After fusion, the fusedcells were plated into six 24-well plates and cultured in α-MEM mediumsupplemented with 10% FBS for 24 hours. The medium was then exchangedwith medium containing 0.7 mg/ml of G418. After growth in the presenceof the G418 antibiotic for about two weeks, the G418 resistant, fusedcells were selected. These G418-resistant clones were used for nucleartransfer, as described below.

[0206] Similarly, ΔΔHAC from the CHO clone ΔΔC13 was transferred intobovine fetal fibroblasts by means of MMCT. The selected G418-resistantclones were used for nuclear transfer.

[0207] Nuclear Transfer, Activation, and Embryo Culture

[0208] The nuclear transfer procedure was carried out essentially asdescribed earlier (Cibelli et al., Science 1998: 280:1256-1258). Invitro matured oocytes were enucleated about 18-20 hours post maturation(hpm) and chromosome removal was confirmed by bisBenzimide (Hoechst33342, Sigma) labeling under UV light. These cytoplast-donor cellcouplets were fused, by using single electrical pulse of 2.4 kV/cm for20 μsec (Electrocell manipulator 200, Genetronics, San Diego, Calif.).After 3-4 hrs, a random sub-set of 25% of the total transferred coupletswas removed, and the fusion was confirmed by bisBenzimide labeling ofthe transferred nucleus. At 30 hpm reconstructed oocytes and controlswere activated with calcium ionophore (5 μM) for 4 minutes (Cal Biochem,San Diego, Calif.) and 10 μg Cycloheximide and 2.5 μg Cytochalasin D(Sigma) in ACM culture medium for 6 hours as described earlier (Lin etal., Mol. Reprod. Dev. 1998: 49:298-307; Presicce et al., Mol. Reprod.Dev. 1994:38:380-385). After activation eggs were washed in HEPESbuffered hamster embryo culture medium (HECM-Hepes) five times andplaced in culture in 4-well tissue culture plates containing irradiatedmouse fetal fibroblasts and 0.5 ml of embryo culture medium covered with0.2 ml of embryo tested mineral oil (Sigma). Twenty five to 50 embryoswere placed in each well and incubated at 38.5° C. in a 5% CO₂ in airatmosphere. On day four 10% FCS was added to the culture medium.

[0209] Embryo Transfer

[0210] Day 7 and 8 nuclear transfer blastocysts were transferred intoday 6 and 7 synchronized recipient heifers, respectively. Recipientanimals were synchronized using a single injection of Lutalyse(Pharmacia & Upjohn, Kalamazoo, Mich.) followed by estrus detection. Therecipients were examined on day 30 and day 60 after embryo transfer byultrasonography for the presence of conceptus and thereafter every 30days by rectal palpation until 270 days. The retention of a HAC in thesebovine fetuses is summarized in Table 3 and is described in greaterdetail in the sections below. TABLE 3 Summary of HAC retention in bovinefetuses HAC Recip/ Reten- Cell Fetus Recovery tion HAC Clone No. NT DateDate Fetal Age H L ΔΔ 4-12 5580 2/14 4/13  58 + + ΔΔ 2-14 5848 2/15 4/13 57 − − ΔΔ 4-12  ^( 5868A) 2/14 6/13 119 + + ΔΔ 4-12  ^( 5868B) 2/146/13 119 + + ΔΔ 4-12  ^( 5542A) 2/14 5/16  91 + + ΔΔ 4-12  ^( 5542B)2/14 5/16  91 + + ΔΔ 4-12 5174 2/14 5/16  91 (abnormal) nd nd ΔΔ 4-126097 2/14 Remains 160 (7/24) nd nd Δ 4-8  6032 1/31 3/30  58 + + Δ 2-135983 2/2  3/30  56 − − Δ 4-2  5968 2/2  3/30  56 + + Δ 2-22 6045 2/2 3/30  56 + + Δ 4-8  5846 1/31 4/20  79 − − Δ 2-13 6053 2/2  4/27  84 + −Δ 4-2  5996 2/1  4/20  77 + −

[0211] Introduction of a HAC Containing a Fragment of Human Chromosome#14

[0212] The SC20 fragment, a human chromosome #14 fragment (“hchr.14fg”,containing the Ig heavy chain gene), was introduced into fetalfibroblast cells in substantially the same manner as described above.Any other standard chromosome transfer method may also be used to insertthis HAC or another HAC containing a human Ig gene into donor cells. Theresulting donor cells may be used in standard nuclear transfertechniques, such as those described above, to generate transgenicungulates with the HAC.

[0213] The pregnancy status of the 28 recipients to whom cloned embryoswere transferred from cells containing the hchr. 14fg was checked byultrasonography. The results are summarized in Table 4. TABLE 4Pregnancy at 40 days using donor cells containing hchr.14fg No of recipsPregnancy at 40 days Clone ID transferred (%) 2-1 08 03 (38) 4-2 10 00(00) 4-1 05 00 (00) 4-1 03 01 (33) 2-1 02 01 (50) Total 28 05 (18)

[0214] The pregnancy rates were lower than anticipated. This is believedto be attributable to extremely abnormally hot weather during embryotransfer.

[0215] As illustrated in FIG. 27, pregnancy rates for HAC carryingembryos appear to be equivalent to non-transgenic cloned pregnancies.One recipient carrying a ΔΔHAC calf gave birth recently to a livehealthy calf. Others will be born over the next several months

[0216] Demonstration of Rearrangement and Expression of Human HeavyChain Locus in a ΔHAC Bovine Fetus

[0217] Cloned ΔHAC-transgenic bovine fetuses were removed at variousgestational days and analyzed for the presence, rearrangement, andexpression of the human immunoglobulin loci. Analysis of genomic DNA andcDNA obtained by RT-PCR from spleen and nonlymphoid tissues (liver andbrain) of one of these fetuses indicated the presence, rearrangement,and expression of the ΔHAC.

[0218] Presence of Human Heavy and/or Light Chain in ΔHAC Fetuses

[0219] To determine whether the human heavy and light chains wereretained in ΔHAC fetuses, liver DNA was isolated from ΔHAC fetuses andanalyzed by PCR for the presence of genomic DNA encoding human heavy andlight chains.

[0220] For the detection of genomic heavy chain DNA, the followingprimers were used: VH3-F 5′-AGTGAGATAAGCAGTGGATG-3′ (SEQ ID NO: 1) andVH3-R 5′-CTTGTGCTACTCCCATCACT-3′ (SEQ ID NO: 2). The primers used fordetection of lambda light chain DNA were IgL-F5′-GGAGACCACCAAACCCTCCAAA-3′ (SEQ ID NO: 3) and IgL-R5′-GAGAGTTGCAGAAGGGGTYGACT-3′ (SEQ ID NO: 4). The PCR reaction mixturescontained 18.9 μl water, 3 μl of 10× Ex Taq buffer, 4.8 μl of dNTPmixture, 10 pmol forward primer and 10 pmol of reverse primer, 1 μl ofgenomic DNA, and 0.3 μl of Ex Taq. Thirty-eight cycles of PCR wereperformed by incubating the reaction mixtures at the followingconditions: 85° C. for three minutes, 94° C. for one minute, 98° C. for10 seconds, 56° C. for 30 seconds, and 72° C. for 30 seconds.

[0221] As shown in FIG. 5, fetuses #5968, 6032 and 6045 each containedboth human heavy chain (μ) and light chain (λ) loci. Fetus #5996contained only the human heavy chain locus. Fetus #5983 did not containthe human heavy chain and may not have contained the human light chain.Fetus #5846 did not contain either human sequence. Thus, fetuses #5983and 5846 may not have retained the HAC. These results suggested thatΔHAC can be stably retained up to gestational day 58 in bovines.

[0222] Presence of Human Cmu Exons in ΔHAC Fetus #5996

[0223] Primers specific for a mRNA transcript including portions of Cmu3 and Cmu 4 were used to determine whether ΔHAC was present andexpressing transcripts encoding the constant region of the human mulocus of fetus #5996.

[0224] For this RT-PCR analysis of the genomic constant region of thehuman mu heavy chain, primers “CH3-F1” (5′accacctatgacagcgtgac-3′, SEQID NO: 5) and “CH4-R2” (5′-gtggcagcaagtagacatcg-3′, SEQ ID NO: 6) wereused to generate a RT-PCR product of 350 base pairs. This PCRamplification was performed by an initial denaturing incubation at 95°C. for five minutes. Then, 35 cycles of denaturation, annealing, andamplification were performed by incubation at 95° C. for one minute, 59°C. for one minute, and 72° C. for two minutes. Then, the reactionmixtures were incubated at 72° C. for 10 minutes. Rearranged bovineheavy chain was detected using primers 17L and P9, as described below(FIG. 7). As an internal control, levels of GAPDH RNA was detected usingprimers “GAPDH forward” (5′-gtcatcatctctgccccttctg-3′, SEQ ID NO: 7) and“GAPDH reverse” (5′-aacaacttcttgatgtcatcat-3′, SEQ ID NO: 8). For thisamplification of GAPDH RNA, samples were incubated at 95° C. for fiveminutes, followed by 35 cycles of incubation at 95° C. for one minute,55° C. for one minute, and 72° C. for two minutes. Then, the mixtureswere incubated at 72° C. for seven minutes.

[0225] This analysis showed that RT-PCR analysis of the spleen of fetus#5996 produced a band (lane 3) matching the amplification productsgenerated using control human spleen cDNA (lane 4) and cDNA obtainedfrom a ΔHAC chimeric mouse (lane 5) (FIG. 6). No such band was detectedin nonlymphoid tissues: bovine liver (lane 1) or bovine brain (lane 2).The capacity of these tissues to support RT-PCR was shown by thesuccessful amplification of the housekeeping gene, GADPH, in both liver(lane 10 of FIG. 6) and brain (lane 6 of FIG. 7).

[0226] Rearrangement of Bovine Heavy Chain Locus by 77 Gestational Days

[0227] The ΔHAC fetus #5996 was tested to determine whether it hadundergone the developmental processes necessary for the expression andactivation of the recombination system required for immunoglobulin heavychain locus rearrangement. For this analysis, standard RT-PCR analysiswas performed to detect the presence of mRNA transcripts encoding mu-VHrearrangements. RNA isolated from the spleen, liver, and brain of fetus#5996 was analyzed by RT-PCR using primers “17L”(5′-ccctcctctttgtgctgtca-3′, SEQ ID NO: 9) and “P9”(5′-caccgtgctctcatcggatg-3′, SEQ ID NO: 10). The PCR reaction mixtureswere incubated at 95° C. for 3 minutes, and then 35 cycles ofdenaturation, annealing, and amplification were performed using thefollowing conditions: 95° C. for one minute, 58° C. for one minute, and72° C. for two minutes. The reaction mixture was then incubated at 72°C. for 10 minutes.

[0228] Lane 5 of FIG. 7 shows that a product of the size expected foramplification of a rearranged bovine heavy chain (450 base pairs) wasobtained. This product migrated to a position equivalent to that of acontrol bovine Cmu heavy chain cDNA known to contain sequencescorresponding to rearranged bovine heavy chain transcripts (lane 7). Asexpected, the rearranged heavy chain was expressed in the spleen (lane5), but absent from the brain (lane 2) and liver (lane 3) at this pointin development.

[0229] Rearrangement and Expression of the Human Heavy Chain Locus inthe ΔHAC Fetus #5996

[0230] The rearrangement and expression of the human heavy chain locuswas demonstrated by the amplification of a segment of DNA includingportions of Cmu and VH regions. Primers specific for RNA transcriptsincluding portions of Cmu (Cmu1) and VH (VH3-30) were used to determineif RNA transcripts containing rearranged human Cmu-VDJ sequences werepresent (FIG. 8).

[0231] For this RT-PCR analysis, primers “Cmu1”(5′-caggtgcagctggtggagtctgg-3′, SEQ ID NO: 11) and “VH3-30”(5′caggagaaagtgatggagtc-3′, SEQ ID NO: 12) were used to produce a RT-PCRproduct of 450 base pairs. This RT-PCR was performed by incubatingreaction mixtures at 95° for 3 minutes, followed by 40 cycles ofincubation at 95° for 30 minutes, 69° for 30 minutes, and 72° for 45minutes, and one cycle of incubation at 72° for 10 minutes. This RT-PCRproduct was then reamplified with the same primers by one cycle ofincubation at 95° C. for three minute, 40 cycles of incubation at 95° C.for one minute, 59° C. for one minute, 72° C. for one minute, and onecycle of incubation at 72° C. for 10 minutes. As an internal control,RT-PCR amplification of GAPDH was performed as described above.

[0232] The gel in FIG. 8 shows that RT-PCR analysis of the spleen fromfetus #5996 produced a band (lane 5) matching the amplification productsgenerated using human spleen cDNA (lane 4) or ΔHAC chimeric mouse spleencDNA (lane 1). No such band was detected in bovine liver (lane 2) orbovine brain (lane 3). As a positive control, amplification of GADPH RNA(lanes 8 and 9) showed the capacity of these tissues to support RT-PCR.

[0233] Rearrangement and expression of the human heavy chain region infetus #5996 was also demonstrated by RT-PCR analysis using primersCH3-F3 (5′-GGAGACCACCAAACCCTCCAAA-3′, SEQ ID NO: 13) and CH4-R2(5′-GTGGCAGCAAGTAGACATCG-3′, SEQ ID NO: 14). These PCR reaction mixturescontained 18.9 μl water, 3 μl of 10× Ex Taq buffer, 4.8 μl of dNTPmixture, 10 pmol forward primer, 10 pmol of reverse primer, 1 μl ofcDNA, and 0.3 μl of Ex Taq. Forty PCR cycles were performed byincubating the reaction mixtures under the following conditions: 85° C.for three minutes, 94° C. for one minute, 98° C. for 10 seconds, 60° C.for 30 seconds, and 72° C. for 30 seconds.

[0234] As shown in lanes 6 and 7 of FIG. 9, an amplified sequence fromthe spleen of fetus #5996 was the same size as the spliced constantregion fragments from the two positive controls: a sample from a humanspleen (lane 8) and a ΔHAC chimeric mouse spleen (lane 9). As expected,the negative controls from a normal mouse spleen and a bovine spleen didnot contain an amplified sequence (lanes 1 and 2). Samples from theliver and brain of fetus #5996 did not contain an amplified splicedsequence of the same size as the spliced human mu heavy chain constantregion fragments but did contain a amplified sequence of an unsplicedgenomic fragment derived from genomic DNA contaminating the RNA sample(lanes 3, 4, and 5).

[0235] VDJ Rearrangement of the Human Heavy Chain Locus in a ΔHAC Fetus

[0236] RT-PCR analysis was also performed to further demonstrate VDJrearrangement in the heavy chain locus in ΔHAC fetus #5996. NestedRT-PCR was performed using primer Cmu-1 (5′-CAGGAGAAAGTGATGGAGTC-3′, SEQID NO: 15) for the first reaction, primer Cmu-2(5′-AGGCAGCCAACGGCCACGCT-3′, SEQ ID NO: 16) for the second reaction, andprimer VH3-30.3 (5′-CAGGTGCAGCTGGTGGAGTCTGG-3′, SEQ ID NO: 17) for bothreactions. The RT-PCR reaction mixtures contained 18.9 μl water, 3 μl of10× Ex Taq buffer, 4.8 μl of dNTP mixture, 10 pmol forward primer, 10pmol of reverse primer, 1 μl of cDNA, and 0.3 μl of Ex Taq. The RT-PCRwas performed using 38 cycles under the following conditions for thefirst reaction: 85° C. for three minutes, 94° C. for one minute, 98° C.for 10 seconds, 65° C. for 30 seconds, and 72° C. for 30 seconds. Forthe second reaction, 38 cycles were performed under the followingconditions: 85° C. for three minutes, 94° C. for one minute, 98° C. for10 seconds, 65° C. for 30 seconds, and 72° C. for 30 seconds usingprimers VH3-30.3 and Cmu-2 (5′-AGGCAGCCAACGGCCACGCT-3′, SEQ ID NO: 16).

[0237] As shown in lanes 6 and 7 of FIG. 10, RT-PCR analysis of thespleen of fetus #5996 produced a heavy chain band of the same size asthe positive controls in lanes 8 and 9. Samples from the liver and brainof fetus #5996 contained some contaminating rearranged DNA (lanes 3 and5). The negative controls in lanes 1 and 2 produced bands of theincorrect size.

[0238] Verification of ΔHAC Rearrangement By Sequencing

[0239] The cDNA obtained by reverse transcription of RNA from the spleenof the ΔHAC fetus #5996 was amplified with primers specific forrearranged human mu and run on an agarose gel. The band produced byamplification with the Cmu1-VH3-30 primer pair was excised from the gel.The amplified cDNA was recovered from the band and cloned. DNA from aresulting clone that was PCR-positive for rearranged human mu waspurified and sequenced (FIG. 11A).

[0240] The sequence from this ΔHAC fetus is greater than 95% homologousto over 20 known human heavy chain sequences. For example, the mu chainof a human anti-pneumococcal antibody is 97% homologous to a region ofthis sequence (FIG. 11B).

[0241] Additional sequences from rearranged human heavy chains were alsoobtained by RT-PCR analysis of the spleen of fetus #5996 using primersCmu-1 and VH3-30.3, followed by reamplification using primers Cmu-2 andVH3-30.3. The RT-PCR products were purified using CHROMA SPIN column(CLONETECH) and cloned into the pCR2.1 TA-cloning vector (Invitrogen)according to manufacturer's protocol. The Dye Terminator sequencereaction (ABI Applied System) was performed in a 10 μl volume reactionmixture composed of BigDye Terminator reaction mixture (3 μl), templateplasmid (200 ng), and the Cmu-2 primer (1.6 pmol). The sequencingreaction was performed using a ABI 3700 sequencer. For this analysis,twenty-five cycles were conducted under the following conditions: 96° C.for one minute, 96° C. for10 seconds, 55° C. for five seconds, and 60°C. for four minutes.

[0242] At least two rearranged human heavy chain transcripts wereidentified, which were VH3-11/D7-27/JH3/Cμ and VH3-33/D6-19/JH2/Cμ(FIGS. 12A and 12B). These results demonstrate that VDJ rearrangement ofthe human mu heavy chain locus occurs in the ΔHAC in the spleen of fetus#5996. The identification of more than one rearranged heavy chainsequence from the same fetus also demonstrates the ability of ΔHACfetuses to generate diverse human immunoglobulin sequences.

Rearrangement and Expression of Human Heavy and Light Chain Loci inΔΔHAC Fetus

[0243] Cloned fetuses derived from bovine fetal fibroblaststranschromosomal for the ΔΔHAC were removed from recipient cows atvarious gestational days. The fetuses were analyzed for the presence andrearrangement of the HAC-borne human immunoglobulin heavy and lambdalight chain loci. Studies of genomic DNA from these tissues indicatedthe presence of the human immunoglobulin heavy and light chains in someof the fetuses. Examination of cDNA derived from the spleens of thesefetuses indicated rearrangement and expression of the immunoglobulinheavy and light chain loci in some of these fetuses. FACS analysis alsodemonstrated the expression of human lambda light chain protein on thesurface of splenic lymphocytes in two of the fetuses.

[0244] Presence of Human Heavy and Light Chain Loci in ΔΔHAC Fetuses Todetermine whether ΔΔHAC fetuses retained the human heavy and light chainloci, PCR analysis was performed on genomic DNA from the liver of 58 dayfetus #5580, 57 day fetus # 5848, and 91 day fetuses #5442A and 5442B.The PCR primers used for detection of the heavy chain loci were VH3-F(5′-AGTGAGATAAGCAGTGGATG-3′, SEQ ID NO: 18) and VH3-R(5′-CTTGTGCTACTCCCATCACT-3′, SEQ ID NO: 19), and the primers used forthe detection of the light chain were IgL-F(5′-GGAGACCACCAAACCCTCCAAA-3′, SEQ ID NO: 20) and IgL-R(5′-GAGAGTTGCAGAAGGGGTYGACT-3′, SEQ ID NO: 21). The PCR reactionmixtures contained 18.9 μl water, 3 ul of 10× Ex Taq buffer, 4.8 μl ofdNTP mixture, 10 pmol forward primer, 10 pmol of reverse primer, 1 μl ofgenomic DNA, and 0.3 ul of Ex Taq. Thirty-eight PCR cycles wereperformed as follows: 85° C. for three minutes, 94° C. for one minute,98° C. for 10 seconds, 56° C. for 30 seconds, and 72° C. for 30 seconds(FIGS. 13 and 14).

[0245] As illustrated in FIGS. 13 and 14, positive control 58 day fetus#5580 contained both human heavy and light chain immunoglobulin loci.Additionally, the 91 day fetuses #5442A and 5442B also contained bothheavy and light chain loci (FIG. 14). In contrast, fetus #5848 did notcontain either human loci and may not have contained ΔΔHAC. Theseresults suggested that ΔΔHAC can be stably retained up to gestationalday 91 in bovine.

[0246] Rearrangement and Expression of Human Heavy Chain Locus in ΔΔHACFetus #5442Δ

[0247] RT-PCR was used to detect expression of rearranged human heavychain RNA transcripts in ΔΔHAC fetus #5542A. The RT-PCR primers usedwere CH3-F3 (5′-GGAGACCACCAAACCCTCCAAA-3′, SEQ ID NO: 22) and CH4-R2(5′-GAGAGTTGCAGAAGGGGTGACT-3′, SEQ ID NO: 23). The RT-PCR reactionmixtures contained 18.9 μl water, 3 μl of 10× Ex Taq buffer, 4.8 μl ofdNTP mixture, 10 pmol forward primer, 10 pmol of reverse primer, 1 μl ofcDNA, and 0.3 μl of Ex Taq. Forty cycles of RT-PCR cycles were performedas follows: 85° C. for three minutes, 94° C. for one minute, 98° C. for10 seconds, 60° C. for 30 seconds, and 72° C. for 30 seconds.

[0248] Lanes 4 and 5 of FIG. 15 contained amplified spliced mu heavychain constant region sequences from the spleen of fetus #5442A that aresimilar in size to that of the positive control samples. These resultsindicate that fetus #5442A expressed a rearranged mu heavy chaintranscript in its spleen. Faint bands were also seen in the region ofthe unspliced genomic sequence, which are amplified from genomic DNAcontaminated in the RNA sample. Control samples from the liver and brainof fetus #5442A did not produce a band of the size expected for anamplified rearranged heavy chain sequence.

[0249] Rearrangement and Expression of Human Heavy Chain Locus in ΔΔHACFetus #5868A

[0250] RT-PCR was used to detect expression of rearranged human heavychain RNA transcripts in the spleen of a ΔΔHAC fetus at 119 gestationaldays (fetus #5868A). The primers used for this analysis were VH30-3(5′-caggtgcagctggtggagtctgg-3′, SEQ ID NO: 24) and CM-1(5′-caggagaaagtgatggagtc-3′, SEQ ID NO: 25). Additionally, primers“GAPDH up” (5′-gtcatcatctctgccccttctg-3′, SEQ ID NO: 26) and “GAPDHdown” (5′-aacaacttcttgatgtcatcat-3′, SEQ ID NO: 27) were used to amplifyGAPDH control transcripts. For this PCR analysis, the reaction mixturewas incubated at 95° C. for five minutes and then multiple cycles ofdenaturation, annealing, and amplification were performed by incubationat 95° C. for one minute, 58° C. for one minute, and 72° C. for twominutes. Then, the mixture was incubated at 72° C. for 10 minutes.

[0251] Lane 3 of FIG. 16 contains the RT-PCR product produced from thisanalysis of ΔΔHAC fetus# 5868A. This RT-PCR product was the sizeexpected for the amplification of a rearranged human heavy chain (470base pairs) and migrated to the same position in the gel as the controlcDNA known to contain sequences corresponding to rearranged human heavychain transcripts. As controls, both ΔΔHAC fetus# 5868A fetal spleencDNA and normal bovine cDNA samples generated a product when amplifiedwith GAPDH primers, demonstrating the capacity of the cDNA to supportamplification (lanes 7 and 8).

[0252] Rearrangement and Expression of Human Lambda Locus in ΔΔHACFetuses #5442A and 5442B

[0253] Primers specific for amplification of a transcript includingportions of human lambda were used to detect RNA transcripts from arearranged human lambda light chain locus.

[0254] For the RT-PCR analysis shown in FIG. 17, an equimolar mixture ofprimers Cλ1 (.5′-GGGAATTCGGGTAGAAGTTCACTGATCAG-3′, SEQ ID NO: 28), Cλ2-3(5′-GGGAATTCGGGTAGAAGTCACTTATGAG-3′, SEQ ID NO: 29), and Cλ7(5′-GGGAATTCGGGTAGAAGTCACTTACGAG-3′, SEQ ID NO: 30) was used with primerVλ1 LEA1 (5′-CCCCCAAGCTTRCCKGSTYYCCTCTCCTC-3′, SEQ ID NO: 31). TheRT-PCR reaction mixtures contained 18.9 μl water, 3 μl of 10× Ex Taqbuffer, 4.8 μl of dNTP mixture, 10 pmol forward primer, 10 pmol ofreverse primer, 1 μl of cDNA and 0.3 μl of Ex Taq. The RT-PCR conditionswere as follows: 40 cycles of 85° C. for three minutes, 94° C. for oneminute, 98° C. for 10 seconds, 60° C. for 30 seconds, and 72° C. for oneminute.

[0255] As shown in FIG. 18, this RT-PCR analysis was also performedusing an equimolar mixture of primers Vλ3LEA1(5′-CCCCCAAGCTTGCCTGGACCCCTCTCTGG-3′; SEQ ID NO:32), Vλ3JLEAD(5′-ATCGGCAAAGCTTGGACCCCTCTCTGGCTCAC-3′, SEQ ID NO: 33), VλBACK4(5′-CCCCCAAGCTTCTCGGCGTCCTTGCTTAC-3′, SEQ ID NO: 34) and an equimolarmixture of primers Cλ1 (5′-GGGAATTCGGGTAGAAGTTCACTGATCAG-3′, SEQ ID NO:35) Cλ2-3 (5′-GGGAATTCGGGTAGAAGTCACTTATGAG-3′, SEQ ID NO: 36) and Cλ7(5′-GGGAATTCGGGTAGAAGTCACTTACGAG-3′, SEQ ID NO: 37). The RT-PCR reactionconditions were the same as those described above for FIG. 7.

[0256] Lanes 6 and 7 of FIG. 17 and lanes 4 and 5 of FIG. 18 containedRT-PCR products from the spleen of fetus #5442A that are similar in sizeto the positive control bands, indicating the presence of rearrangedlight chain RNA transcripts in this fetus. The spleen sample from fetus#5442B produced very weak bands of the appropriate size which are notvisible in the picture. This RT-PCR product indicates that fetus #5442Balso expressed a rearranged light chain immunoglobulin transcript in itsspleen. As expected, samples from the brain of fetuses #5442A and 5442Bdid not express human rearranged lambda light chain transcripts.

[0257] Rearrangement and Expression of Human Lambda Locus in ΔΔHAC Fetus#5868A

[0258] RNA transcripts from a rearranged human lambda light chain locuswere also detected in ΔΔHAC fetus# 5868A. For this analysis, primersspecific for amplification of a transcript including portions of humanlambda were used to detect ΔΔHAC-encoded expression of transcriptsencoding portions of a rearranged human lambda locus. Primer VL1 LEAI(5′-cccccaagcttRccKgStYYcctctcctc-3′; SEQ ID NO:38) and an equimolarmixture of primers CL1 (5′-gggaattcgggtagaagtcactgatcag-3′; SEQ IDNO:39), CL2-3 (5′-gggaattcgggtagaagtcacttatgag-3′; SEQ ID NO:40), andCL7 (5′-gggaattcgggtagaagtcacttacgag-3′; SEQ ID NO:41) were used forthis analysis. For this RT-PCR reaction, the reaction mixtures wereincubated at 95° C. for 5 minutes and then multiple cycles ofdenaturation, annealing, and amplification were performed by incubationat 95° C. for one minute, 60° C. for one minute, and 72° C. for twominutes. Then, the mixtures were incubated at 72° C. for 10 minutes.

[0259] This analysis demonstrated that spleen cDNA from ΔΔHAC #5868A(lane 4 of FIG. 19) produced a RT-PCR product of the same size as the TCmouse spleen cDNA (lane 6) positive control. No such RT-PCR product wasdetected using either brain or liver cDNA from ΔΔHAC #5868A (lanes 2 and3, respectively). The capacity of each of these tissues to supportRT-PCR was shown by successful amplification of the housekeeping gene,GAPDH using primers “GAPDH up” and “GAPDH down” (lanes 8 and 10).

[0260] Verification of ΔΔHAC Rearrangement by Sequencing

[0261] RT-PCR analysis was performed on a spleen sample from fetus#5442A using an equimolar mixture of primers Cλ1, Cλ2-3, and Cλ7 withprimer Vλ1LEA1, or an equimolar mixture of primersVλ3LEA1, Vλ3JLEAD, andVλBACK4 and an equimolar mixture of primers Cλ1, C/2-3, and Cλ7 in. ThePCR products were purified using a CHROMA SPIN column (CLONETECH) andcloned into the pCR2.1 TA-cloning vector (Invitrogen), according tomanufacturer's protocol. The Dye Terminator sequence reaction (ABIApplied System) was carried out using the Cλ1, Cλ2-3, and Cλ7 primers inan equimolar mixture. Twenty-five cycles were performed at 96° C. forone minute, 96° C. for 10 seconds, 55° C. for five seconds, and 60° C.for four minutes. The 10 μl reaction mixture contained BigDye Terminatorreaction mixture (3 μl), template plasmid (200 ng), and the Cλ1, Cλ2-3,and Cλ7 primers (1.6 pmol). The reaction mixture was analyzed using aABI 3700 sequencer.

[0262] At least two rearranged human lambda light chain transcripts wereidentified (V1-17/JL3/Cλ and V2-13/JL2/Cλ). These results demonstratethat VJ rearrangement of human lambda light chain genes occurs in theΔΔHAC in the spleen of fetus #5442A (FIGS. 20 and 21).

[0263] FACS Analysis of Expression of Human Lambda Light Chain andBovine Heavy Chain in ΔΔHAC Fetus #5442A and 5442B

[0264] Splenic lymphocytes from ΔΔHAC Fetus #5442A and 5442B wereanalyzed for the expression of human lambda light chain and bovine heavychain proteins. These cells were reacted with a phycoerytherin labeledanti-human lambda antibody (FIGS. 22C and 22D), a FITC labeledanti-bovine IgM antibody (FIGS. 22D and 22H), or no antibody (FIGS. 22A,22B, 22E, and 22F) for 20 minutes at 4° C. Cells were then washed twicewith PBS plus 2% FCS and analyzed on a FASCalibur cell sorter. Thepercent of cells reacting with the antibody was calculated using the nonantibody controls to electronically se the gates. These percentages aredisplayed beneath each histogram. Fetus # 5442A (FIGS. 22A-22D) andfetus #5442B (FIGS. 22E-22H) expressed both human lambda light chainprotein and bovine heavy chain protein.

EXAMPLE 3 Transgenic Ungulates Producing Xenogenous Antibodies andReduced Amounts of Endogenous Antibodies

[0265] Transgenic ungulates expressing a xenogenous antibody and havinga reduced level of expression of endogenous antibodies may also begenerated. By increasing the number of functional xenogenousimmunoglobulin heavy or light chain genes relative to the number offunctional endogenous heavy or light chain genes, the percentage of Bcells expressing xenogenous antibodies should increase.

[0266] To generate these transgenic ungulates, ΔHAC or ΔΔHAC transgenicungulates may be mated with transgenic ungulates containing a mutationin one or both alleles of an endogenous immunoglobulin chain (e.g., a muheavy chain or a lambda or kappa light chain). If desired, the resultingtransgenic ungulates may be mated with (i) transgenic ungulatescontaining a mutation in one or both alleles of an endogenousalpha-(1,3)-galactosyltransferase, prion, and/or J chain nucleic acid or(ii) transgenic ungulates containing an exogenous J chain nucleic acid(e.g., human J chain). Alternatively, a cell (e.g., a fetal fibroblast)from a ΔHAC or ΔΔHAC transgenic fetus may be genetically modified by themutation of one or more endogenous immunoglobulin genes. In anotherpossible method, ΔHAC or ΔΔHAC is introduced into a cell (e.g., a fetalfibroblast) in which endogenous immunoglobulins (mu heavy and/or lambdalight chains) are hemizgously or homozygously inactivated. In any of theabove methods, the cells may also be genetically modified by (i) theintroduction of a mutation, preferably a knockout mutation, into one orboth alleles of an endogenous alpha-(1,3)-galactosyltransferase, prion,and/or J chain nucleic acid or (ii) the introduction of an exogenous Jchain nucleic acid. The resulting transgenic cell may then be used innuclear transfer procedures to generate the desired transgenicungulates. Exemplary methods are described below.

[0267] DNA Constructs

[0268] The mu heavy chain (FIG. 2A), lambda light chain, kappa lightchain, alpha-(1,3)-galactosyltransferase, prion, and/or J chain knockoutconstructs described above may be used. Alternatively, the puromycinresistant mu heavy chain construct described below may be used (FIG.3F). This knockout construct was designed to remove the 4 main codingexons of the bovine mu heavy chain locus but leave the transmembranedomain intact, resulting in the inactivation of the mu heavy chainlocus.

[0269] The puromycin resistant construct was assembled as follows. A 4.4kilobase XhoI fragment containing the region immediately proximal tocoding exon 1 was inserted into the XhoI site of pBluescript II SK+.Plasmid pPGKPuro, which contains a puromycin resistant gene, wasobtained from Dr. Peter W. Laird, Whitehead Institute, USA. A 1.7 KbXhoI fragment containing a puromycin resistance gene was subclonedadjacent to, and downstream of, the 4.4 Kb fragment into the SalI sitepresent in the polylinker region. This 1.7 Kb puromycin marker replacesthe coding exons CH1, CH2, CH3 and CH4 of the bovine immunoglobulinheavy chain locus. An XbaI fragment containing a 4.6 Kb region of the mulocus that is downstream of these four exons in the wild-type genomicsequence was added to this construct for use as the second region ofhomology.

[0270] To generate the final targeting construct, a subclone of thisconstruct was generated by cutting the three assembled fragments withNotI and MluI The MluI restriction digestion truncates the 4.6 Kbfragment down to 1.4 Kb. The NotI site lies in the polylinker and doesnot cut into the subcloned DNA itself. The MluI site was filled in witha Klenow fragment to generate a blunt end, and the NotI/filled in MluIfragment was subcloned into a fresh pBluescript II SK+vector using theNotI and Smal sites present in the pBluescript vector. For genetargeting, the final vector is linearized with NotI.

[0271] Gene Targeting by Electroporation and Drug Selection ofTransfected Fibroblasts

[0272] For electroporation, a single cell suspension of 1×10⁷ bovinefetal fibroblasts (e.g, fibroblasts obtained as described in Example 2from a ΔHAC or ΔΔHAC transgenic fetus) that had undergone a limitednumber of population doublings is centrifuged at 1200 rpm for fiveminutes and re-suspended in 0.8 ml of serum-free Alpha-MEM medium. There-suspended cells are transferred to a 0.4 cm electroporation cuvette(Invitrogen, cat#. P460-50). Next, 30 μg of a restrictionenzyme-linearized, gene targeting vector DNA is added, and the contentsof the cuvette are mixed using a 1 ml pipette, followed by a two minuteincubation step at room temperature. The cuvette is inserted into theshocking chamber of a Gene Pulser II electroporation system (Biorad) andthen electroporated at 1000 volts and 50 μF. The cuvette is quicklytransferred to a tissue culture hood and the electroporated cells arepipetted into approximately 30 ml of complete fibroblast medium. Thecells are equally distributed into thirty 100 mm tissue culture dishes(Corning, cat#. 431079), gently swirled to evenly distribute the cells,and incubated at 38.5° C./5% CO₂ for 16 to 24 hours The media is removedby aspiration and replaced with complete fibroblast medium containingthe selection drug of choice. The media is changed every two days andcontinued for a total time period of 7 to 14 days. During the drugselection process, representative plates are visually monitored to checkfor cell death and colony formation. Negative control plates are set upthat contained fibroblasts that are electroporated in the absence of thegene targeting vector and should yield no colonies during the drugselection process.

[0273] Picking of Drug Resistant Fibroblast Colonies and Expansion ofCells

[0274] Following completion of the drug selection step (usually 7 to 14days), the drug resistant colonies are macroscopically visible and readyfor transfer to 48 well tissue culture plates for expansion. To assistin the transferring process, individual colonies are circled on thebottom of the tissue culture plate using a colored marker (Sharpie).Tissue culture plates containing colonies are washed 2× with 1× D-PBS(without Ca²⁺ and Mg²⁺) and then 5 ml of a 1:5 dilution of the celldissociation buffer is added per plates. Following a 3 to five minuteroom temperature incubation step, individual colonies start to detachfrom the bottom of the tissue culture dish. Before the coloniesdetached, they are individually transferred to a single well of a 48well tissue culture plate using a P200 pipetmen and an aerosol barrierpipette tip (200 or 250 μl). Following transfer, the colony iscompletely dissociated by pipeting up-and-down and 1 ml of completefibroblast medium is added. To ensure that the cells are drug resistant,drug selection is continued throughout the 48 well stage. Thetransferred colonies are cultured at 38.5° C./5% CO₂ and visuallymonitored using an inverted microscope. Two to seven days later, wellsthat are approaching confluency are washed two times with 1× D-PBS(without Ca²⁺ and Mg²⁺) and detached from the bottom of the well by theaddition of 0.2 ml of cell dissociation buffer, followed by a fiveminutes room temperature incubation step. Following detachment, thecells are further dissociated by pipeting up-and-down using a P1000pipetmen and an aerosol pipette tip (1000 μl). Approximately 75% of thedissociated fibroblasts are transferred to an individual well of a 24well tissue culture plate to expand further for subsequent PCR analysisand the remaining 25% is transferred to a single well of a second 24well plate for expansion and eventually used for somatic cell nucleartransfer experiments. When cells in the plate containing 75% of theoriginal cells expanded to near confluency, DNA is isolated from thatclone for genetic analysis.

[0275] DNA Preparation

[0276] The procedure used to isolate DNA for genetic analyses is adaptedfrom Laird et al, Nucleic Acids Research, 1991, Volume 19, No. 15. Inparticular, once a particular clone has attained near-confluency in onewell of a 24 well plate, culture medium is aspirated from that well andthe adherent cells are washed twice with PBS. The PBS is aspirated offand replaced with 0.2 ml buffer to lyse the cells and digest excessprotein from the DNA to be isolated. This buffer is composed of 100 mMTris-HCl (pH 8.5), 5 mM EDTA, 0.2% SDS, 200 mM NaCl and 100 ug/mlproteinase K. The 24 well plate is returned to the tissue cultureincubator for a minimum of three hours to allow the release of the DNAand digestion of protein. The viscous product of this procedure istransferred to a 1.5 ml microcentrifuge tube and 0.2 ml of isopropanoladded to precipitate the DNA. The precipitate is recovered bycentrifugation, the DNA pellet is rinsed with 70% ethanol, and afterair-drying, the pellet is resuspended in 25-50 ul of buffer containing10 mM Tris, pH 8, and 1 mM EDTA. This DNA is used for PCR analyses ofclones.

[0277] Screening of Clones

[0278] Two different approaches are used to screen clones, bothemploying the polymerase chain reaction (PCR). All approaches describedin this section are adaptable to the targeting of any other gene, theonly difference being the sequences of the primers used for geneticanalysis.

[0279] According to the first approach, two separate pairs of primersare used to independently amplify products of stable transfection. Onepair of primers is used to detect the presence of the targeting vectorin the genome of a clone, regardless of the site of integration. Theprimers are designed to anneal to DNA sequences both present in thetargeting vector. The intensity of the PCR product from this PCRreaction may be correlated with the number of copies of the targetingvector that have integrated into the genome. Thus, cells containing onlyone copy of the targeting vector tend to result in less intense bandsfrom the PCR reaction. The other pair of primers is designed to detectonly those copies of the vector that integrated at the desired locus. Inthis case, one primer is designed to anneal within the targeting vectorand the other is designed to anneal to sequences specific to the locusbeing targeted, which are not present in the targeting vector. In thiscase, a PCR product is only detected if the targeting vector hasintegrated directly next to the site not present in the targetingvector, indicating a desired targeting event. If product is detected,the clone is used for nuclear transfer.

[0280] For the neomycin resistant heavy chain knockout construct,primers Neol (5′-CTT GAA GAC GAA AGG GCC TCG TGA TAC GCC-3′, SEQ ID NO:42) and IN2521 (5′-CTG AGA CTT CCT TTC ACC CTC CAG GCA CCG-3′, SEQ IDNO: 43) are used to detect the presence of the targeting vector incells, regardless of the location of integration. Primers Neol andOUT3570 (5′-CGA TGA ATG CCC CAT TTC ACC CAA GTC TGT C-3′, SEQ ID NO: 44)are used to specifically amplify only those copies of the targetingconstruct that integrated into the mu heavy chain locus.

[0281] For these PCR reactions to analyze the integration of theneomycin resistant heavy chain knockout construct, a Qiagen PCR kit isused. The PCR reaction mixture contains 1 pmole of each primer, 5 ul of10× reaction buffer, 10 ul of Q solution, 5 ul of DNA, and 1 ul of dNTPsolution. The reaction mixture is brought to a total volume of 50 ulwith H₂O. This PCR amplification is performed using an initialdenaturing incubation at 94° C. for two minutes. Then, 30 cycles ofdenaturation, annealing, and amplification are performed by incubationat 94° C. for 45 seconds, 60° C. for 45 seconds, and 72° C. for twominutes. Then, the reaction mixture is incubated at 72° C. for fiveminutes and at 4° C. until the mixture is removed from the PCR machine.

[0282] In the alternative approach, a single primer set is used toamplify the targeted locus and the size of the PCR products isdiagnostic for correct targeting. One primer is designed to anneal to aregion of the locus not present in the targeting vector and the otherprimer is designed to anneal to a site present in the targeting vectorbut also present in the wild type locus. In this case, there is nodetection of targeting vector that had integrated at undesirable sitesin the genome. Because the region deleted by the targeting vector isdifferent in size from the drug selection marker inserted in its place,the size of the product depended on whether the locus amplified is ofwild-type genotype or of targeted genotype. Amplification of DNA fromclones containing incorrect insertions or no insertions at all of thetargeting vector results in a single PCR product of expected size forthe wild type locus. Amplification of DNA from clones containing acorrectly targeted (“knocked out”) allele results in two PCR products,one representing amplification of the wild type allele and one ofaltered, predictable size due to the replacement of some sequence in thewild-type allele with the drug resistance marker, which is of differentlength from the sequence it replaced.

[0283] For the puromycin resistant heavy chain knockout construct,primers Shortend (5′-CTG AGC CAA GCA GTG GCC CCG AG-3′, SEQ ID NO: 45)and Longend (5′-GGG CTG AGA CTG GGT GAA CAG AAG GG-3′, SEQ ID NO: 46)are used. This pair of primers amplifies both the wild-type heavy chainlocus and loci that have been appropriately targeted by the puromycinconstruct. The size difference between the two bands is approximately0.7 Kb. The presence of the shorter band is indicative of appropriatetargeting.

[0284] For this PCR reaction to analyze the integration of thepuromcying resistant heavy chain knockout construct, a Promega MasterMix kit is used. The PCR reaction mixture contains 1 pmole of eachprimer, 2.5 ul of DNA, and 25 ul of 2× Promega Master Mix. The reactionmixture is brought to a total volume of 50 ul with H₂O. This PCRamplification is performed using an initial denaturing incubation at 94°C. for two minutes. Then, 30 cycles of denaturation, annealing, andamplification are performed by incubation at 94° C. for 45 seconds, 60°C. for 45 seconds, and 72° C. for two minutes. Then, the reactionmixture is incubated at 72° C. for five minutes and at 4° C. until themixture is removed from the PCR machine.

[0285] First Round of Nuclear Transfer

[0286] Selected fibroblast cells in which an immunoglobulin gene hasbeen inactivated may be used for nuclear transfer as described inExample 2 to generate a transgenic ungulate containing a mutation in anendogenous immunoglobulin gene and containing a HAC encoding anxenogenous immunoglobulin gene. Alternatively, nuclear transfer may beperformed using standard methods to insert a nucleus or chromatin mass(i.e., one or more chromosomes not enclosed by a membrane) from aselected transgenic fibroblast into an enucleated oocyte (U.S. Ser. No.60,258,151; filed Dec. 22, 2000). These methods may also be used forcells in which an endogenous alpha-(1,3)-galactosyltransferase, prion,and/or J chain nucleic acid has been mutated.

[0287] Second Round of Mutagenesis and Nuclear Transfer

[0288] If desired, a cell (e.g., a fetal fibroblast) may be obtainedfrom a transgenic ungulate generated from the first round of nucleartransfer. Another round of gene targeting may be performed as describedabove to inactivate the second allele of the gene inactivated in thefirst round of targeting. Alternatively, another immunoglobulin (e.g.,mu heavy chain, lambda light chain, kappa light chain, or J chain),alpha-(1,3)-galactosyltransferase, or prion gene may be inactivated inthis round of targeting. For this second round of targeting, either ahigher concentration of antibiotic may be used or a knockout constructwith a different antibiotic resistance marker may be used. Antibioticresistance cells may be selected as described above. The selected cellsmay be used in a second round of nuclear transfer as described above togenerate, for example, a transgenic ungulate containing two mutations inendogenous immunoglobulin genes and containing a HAC encoding anxenogenous immunoglobulin gene. Alternatively, the selected antibioticresistant cells may first be treated to isolate G1 phase cells asdescribed below, which are used for the second round of nucleartransfer.

[0289] To isolation of G1 cells for nuclear transfer, 5.0×10⁵ cells areplated onto 100 mm tissue culture plates containing 10 ml of α-MEM+FCS,twenty four hours prior to isolation. The following day, plates arewashed with PBS and the culture medium is replaced for 1-2 hours beforeisolation. The plates are then shaken for 30-60 seconds on aVortex-Genie 2 (Fisher Scientific, Houston, Tex., medium speed), themedium is removed, spun at 1000 G for five minutes and the pellet isre-suspended in 250 μl of MEM +FCS. Newly divided cell doublets attachedby a cytoplasmic bridge, are then selected, as these cells are in earlyG1. This isolation procedure is referred to as the “shake off” method.

EXAMPLE 4 Transgenic Ungulates Having Reducedα-1,3-galactosyltransferase Activity

[0290] Bovine fibroblast cell lines in which one allele of theα-1,3-galactosyltransferase locus is mutated were generated byhomologous recombination. The DNA construct for generating theα-galactosyltransferase knockout cells was used to prevent transcriptionof functional, full-length α-galactosyltransferase mRNA by insertingboth a puromycin-resistance gene (puro, described in Example 3) and atranscription termination cassette (STOP) in exon 9 which contains thecatalytic domain. Thus, the resulting immature α-galactosyltransferasetranscripts lack the catalytic domain. The DNA construct (i.e., theα-galactosyltransferase KO vector) was electroporated into threeindependent bovine fibroblast cell lines, and then puromycin-resistantcolonies were isolated. Based on PCR analysis, homologous recombinationin the exon 9 region occurred in some colonies. Thus, bovine fibroblastcell lines in which one allele of α1,3-galactosyltransferase locus ismutated were generated. If desired, the second allele can be mutated byusing the same knockout vector and a higher concentration of antibioticto select for homozygous knockout cells or using another knockout vectorwith a different antibiotic resistance gene. This method may also beapplied to cells from other ungulates to generate transgenic cells foruse in the nuclear transfer methods described herein to producetransgenic ungulates of the present invention.

[0291] These methods are described further below.

[0292] Construction of an α-1,3-galactosyltransferase KO Vector

[0293] The α-1,3-galactosyltransferase KO vector was generated asfollows (FIG. 23). To isolate genomic DNA around exon 9 of theα-1,3-galactosyltransferase gene, a DNA probe was amplified by PCR usingthe following primer pair 5′-gatgatgtctccaggatgcc-3′ (SEQ ID NO: 61) and5′-gacaagcttaatatccgcagg-3′ (SEQ ID NO: 62). Using this probe, a bovinegenomic λ phage library was screened, and 7 positive X phage clones wereidentified. One clone, which contained DNA from a male Charolais bovinefibrolast cell, was analyzed further by restriction mapping. The NotI-Xho I genomic fragment containing exon 9 was subcloned intopBluescript II SK(−), and then both puro and STOP cassettes wereinserted at the Avi I site in the Not I-Xho I genomic fragment which is5′ to the catalytic domain. A diphtheria toxin gene (DT-A, Gibco) wasalso added to the vector construct to kill cells in which the targetingcassette was integrated nonhomologously.

[0294] Transfection/Knockout Procedures

[0295] Transfection of three fetal fibroblasts cell lines (two from amale Jersey bovine and one from a female Jersey bovine) was performedusing a standard electroporation protocol as follows. The medium used toculture the bovine fetal fibroblasts contained 500 ml Alpha MEM (Gibco,12561-049), 50 ml fetal calf serum (Hy-Clone #ABL1 3080), 5 mlpenicillin-streptomycin (SIGMA), and 1 ml 2-mercaptoethanol (Gibco/BRL#21985-023). On the day prior to transfection, cells were seeded on aT175 tissue culture flask with a targeted confluency of 80-100%, asdetermined by microscopic examination. On the day of transfection, about107 bovine fibroblasts cells were trypsinized and washed once withalpha-MEM medium. After resuspension of the cells in 800 μl ofalpha-MEM, 30 μg of DNA was added to the cell suspension and mixed wellby pipetting. The cell-DNA suspension was transferred into anelectroporation cuvette and electroporated at 1,000 V and 50 μF. Afterthat, the electroporated cells were plated onto twenty 24-well plateswith the alpha-MEM medium supplemented with the serum. After a 48hour-culture, the medium was replaced with medium containing 1 μg/ml ofpuromycin, and the cells were cultured for 2-3 weeks to select puromycinresistant cells. After selection, all colonies which reached close to100% confluency were picked, and genomic DNA was extracted from thecolonies to screen for the desired homologous recombination events byPCR.

[0296] Screening for Targeted Integrations

[0297] As described above, the genomic DNA was extracted from each24-well independently using the PUREGENE DNA isolation Kit (GentraSYSTEMS) according to the manufacture's protocol. Each genomic DNAsample was resuspended in 20 μl of 10 mM Tris-Cl (pH 8.0) and 1 mM EDTA(EDTA). Screening by PCR was performed using the following primer pair5′-aagaagagaaaggtagaagaccccaaggac-3′ (SEQ ID NO: 63) and5′-cctgggtatagacaggtgggtattgtgc-3′ (SEQ ID NO: 64). The sequence of oneprimer is located in the α-1,3-galactosyltransferase KO vector, and thesequence of the other primer is located just outside of the integratedvector in the targeted endogenous locus (FIG. 23). Therefore, theexpected PCR product should be detected only when the KO vector isintegrated into the targeted locus by homologous recombination.

[0298] The PCR reaction mixtures contained 18.9 μl water, 3 μl of 10× LAPCR buffer II (Mg²⁺ plus), 4.8 μl of dNTP mixture, 10 pmol forwardprimer, 10 pmol of reverse primer, 1 μl of genomic DNA, and 0.3 μl of LATaq. Forty cycles of PCR were performed by incubating the reactionmixtures at the following conditions: 85° C. for three minutes, 94° C.for one minute, 98° C. for 10 seconds, and 68° C. for 15 minutes. AfterPCR, the reaction mixtures were analyzed by electrophoresis.Puromycin-resistant clones which generated PCR products of the expectedsize were selected (FIG. 23). Thus, bovine fibroblast cell lines inwhich one allele of the α-1,3-galactosyltransferase locus is mutated bythe KO vector were successfully generated.

EXAMPLE 5

[0299] Alternative Method for Producing Transgenic Ungulates usingAdeno-associated Viruses to Mutate an Endogenous Gene Adeno-associatedvirus (AAV) can be used for specific replacement of targeted sequencespresent in the genome of cells (Inoue et al., Mol. Ther. 3(4):526-530,2001); Hirata et al., J Virol. 74(10):16536-42, 2000); Inoue et al., J.Virol. 73(9):7376-80, 1999); and Russell et al., Nat. Genet.18(4):325-30,1998)). The gene targeting rate is highly efficient incomparison to more conventional gene targeting approaches. AAV has abroad range of host and tissue specificities, including specificity forboth bovine and human skin fibroblasts. Thus, AAV can be used to producetransgenic ungulate cells containing one or more mutations in anendogenous immunoglobulin (e.g., mu heavy chain, lambda light chain,kappa light chain, or J chain), alpha-(1,3)-galactosyltransferase, orprion gene. These transgenic cells can then be used in the nucleartransfer methods described herein to produce transgenic ungulates of thepresent invention.

[0300] Using AAV resulted in homologous recombination of the bovineimmunoglobulin heavy chain locus at higher frequencies than previouslyobtained using traditional gene targeting strategies (i.e.,electroporation and lipofection procedures). In the first round of genetargeting experiments, five appropriately targeted fibroblast cloneswere obtained out of 73 stable transductants containing the DNAintroduced through an AAV vector.

[0301] These experiments were carried out as follows.

[0302] AAV Knockout Vectors

[0303] AAV constructs can disrupt a gene either by simple insertion offoreign sequences or replacement of endogenous sequences with newsequence present in the AAV vector. FIG. 24 shows an AAV construct inwhich all four coding exons of the bovine immunoglobulin heavy chain muconstant region are present on a 2822 base pair BamHI-XhoI fragment. A1.16 Kb fragment containing a neomycin resistance marker present in thecommercially available vector, pMC1Neo, was inserted into a SacII sitepresent in exon 4 of the mu heavy chain locus from a Holstein bovine.This locus is the one contained in the phage clone isolated to generatethe knockout vector described in Example 1. To generate the AVV vector,the SacII site in the mu heavy chain locus was filled in to create bluntends, which were then ligated to blunt SalI linkers (New EnglandBiolabs). Then, the XhoI fragment of pMC1Neo, which contains theneomycin resistance gene, was ligated to the SalI site added to thelocus through the Sail linker. This ligation can be performed becausethe XhoI and SalI restriction sites have compatible ends. This knockoutvector causes a disruptional insertion of the neomycin resistance geneinto the endogenous mu heavy chain gene, thereby inactivating the muheavy chain gene. This gene inactivation occurs without deleting regionsof the endogenous mu locus.

[0304] An alternative vector was designed to remove exons 3 and 4 fromthe endogenous locus during targeting, resulting in the replacement ofthese two exons with a functional copy of the neomycin resistance gene(FIG. 25). This construct was generated using PCR amplification ofgenomic DNA from a female Jersey bovine. In particular, the 3′ region ofhomology was amplified using the following primers: 5′GGGGTCTAGAgcagacactacactgatgggcccttggtcc 3′ (SEQ ID NO: 65), which addsa XbaI restriction site, and 5′ GGGGAAGCTTcgtgtccctggtcctgtctgacacag 3′(SEQ ID NO: 66), which adds a HindIII restriction site. The 5′ region ofhomology was amplified with primers 5′GGGGCTCGAGgtcggcgaaggatggggggaggtg 3′ (SEQ ID NO: 67), which adds a XhoIrestriction site, and 5′ GGGGGGTACCgctgggctgagctgggcagagtggg 3′ (SEQ IDNO: 68), which adds a KpnI restriction site. The capitalized nucleotidesin these primer sequences are nucleotides that do not anneal to the muheavy chain locus but are included in the primers to add restrictionsites to facilitate later subcloning steps. The first four guanines areadded to separate the restriction sites from the very end of the primersbecause restriction enzymes do not cleave sites that are at the very endof primers as well as internal sites. The 5′ region of homology is 1.5Kb long and contains exons 1 and 2. The 5′ region of homology alsocontains the first 25 nucleotides of exon 3 to maintain the spliceacceptor site of exon 3. The splice acceptor site allows exon 3 to beused for splicing and thus prevents the possible splicing of exons 1 and2 to the downstream transmembrane domain to form an aberrantmembrane-bound product. The 3′ region of homology is 1.24 Kb long andcontains the region immediately downstream of exon 4.

[0305] For the construct shown in FIG. 24, the targeting cassette wasinserted into the AAV vector reported by Ryan et al. (J. of Virology70:1542-1553, 1996), which contains viral long terminal repeat (LTR)sequences, using standard methods. The AAV vector was packaged intocapsids using the TtetA2 packaging cell line as previously described(Inoue and Russell, 1998, J. Virol. 72:7024-7031, 1998) and purified aspreviously described (Zolotukhin et al., Gene Therapy, 6: 973-985,1999). For the construct shown in FIG. 25, the above method or any otherstandard method can be used to insert the targeting cassette into theAAV vector described by Ryan et aL or any other AAV vector (such as acommercially available vector from Stratagene) and generate virusescontaining the vector.

[0306] Transduction Procedures

[0307] Fibroblasts from a female Jersey bovine were seeded into one wellof a 48 well tissue culture plate at 40,000 cells per well and culturedin complete medium at 38.5° C. and 5% CO₂ until cells attached to thebottom surface of the well. Once cells adhered, the medium was removedand replaced with 0.2 ml of fresh medium containing AAV particles withthe vector shown in FIG. 24 at a multiplicity of infection (MOI) of500-20,000 particles/cell. The MOI was chosen based on pilot experimentsthat determined the resulting numbers of colonies and the spacing of thecolonies during the drug selection phase. Plates were incubatedovernight. After this incubation, the transduced wells were rinsed withcalcium and magnesium-free PBS and detached from the wells using eithertrypsin or the cell dissociation buffer described above. A uniform cellsuspension was obtained by gentle pipetting of the detached cells, andthe cells from the well were redistributed among ten 100 mm tissueculture dishes. Dishes were incubated with complete medium overnight.

[0308] Following this incubation of the 100 mm dishes, the medium wasreplaced with selective medium containing G418 at a concentration of 350micrograms/ml. Selective medium was changed every 2-3 days untilcolonies were macroscopically visible on the surface of the dish. Atthat point, individual colonies were picked and transferred into theirown vessels.

[0309] Regions containing colonies were marked on the outer surface ofthe tissue culture dish. Once all colonies were circled, medium wasaspirated off the plates, and the plates were washed three times withcalcium and magnesium-free PBS. After washing, the plates were floodedwith a 1:25 dilution of 1× trypsin and allowed to sit at roomtemperature until the colonies had visibly begun to detach from thesurface of the plate. Plates were kept stationary to prevent detachedcolonies from floating to another location of the plate. A pipet tip wasused to pick up cell clumps in a volume of 50 microliters, and thecontents of the pipet tip were transferred into one well of a 24 welltissue culture plate. Once all colonies were transferred, completemedium containing G418 was added, and the isolated clones were allowedto proliferate to near confluency.

[0310] When an individual well was close to confluency, it was washedtwice with calcium and magnesium free PBS. Cells were detached using 0.2ml of cell dissociation buffer. Of this cell suspension, 20 μl wastransferred to a new 24 well plate, and the remaining cells were allowedto reattach to the surface of the original 24 well plate following theaddition of 2.0 ml of complete medium. The original plate was incubatedto 100% confluency. The new plate serves as a source of appropriatelytargeted cells for future bovine cloning procedures.

[0311] When a well from the original 24 well plate became 100%confluent, the medium was removed, and the cells were washed once withPBS. PBS was removed and replaced with a cell lysis buffer adopted fromLaird et al. (Nucleic Acids Res. 19:4293, 1991). Briefly, 0.2 ml oflysis buffer containing 200 mM NaCl, 100 mM Tris-HCl pH 8.5, 5 mM EDTA,0.2% SDS, and 100 ug/ml proteinase K was added to the well. The platewas returned to the incubator for between three hours and overnight. Theviscous cell lysate was then transferred to a microfuge tube. An equalvolume of isopropanol was added to precipitate DNA. Following a 10minute spin in a microfuge, the supernatant was discarded, and thepellet was washed once with 0.5 ml of 70% ethanol. After removal of theethanol, the DNA pellet was air-dried and resuspended in 35 microlitersof TE buffer (10 mM Tris pH 8 and 1 mM EDTA). Aliquots of 3 μl were usedfor PCR analysis.

[0312] PCR analysis

[0313] DNA samples from drug resistant clones transduced with AAVparticles were screened for appropriate targeting of the vector usingPCR analysis. This screening strategy used one primer that annealswithin the DNA encoding the drug selection marker and another primerthat anneals within the targeted locus, but outside the sequence presentin the AAV targeting particles. PCR products are only detected if theAAV targeting DNA has integrated into the desired location of theendogenous genome.

[0314] Results from a single targeting experiment using these AAVparticles are shown in FIG. 26. Based on this analysis, five out of 73independent clones contained the appropriate targeted vector DNA.

[0315] This method may also be used with the AAV vector shown in FIG. 25or with any other appropriate adenovirus or adeno-associated viralvector. If desired, the second mu heavy chain allele can be mutated inthe isolated colonies by transducing them with an AAV vector with adifferent antibiotic resistance gene (i.e., a gene other than a neomycinresistance gene). To select the resulting homozygous knockout cells, theinfected cells are cultured in the presence of the correspondingantibiotic. Alternatively, the isolated colonies can be transduced withan AAV vector containing a neomycin resistance gene and cultured in thepresence of a high concentration of antibiotic (i.e., a concentration ofantibiotic that kills heterozygous knockout cells but not homozygousknockout cells).

[0316] Other Embodiments

[0317] From the foregoing description, it will be apparent thatvariations and modifications may be made to the invention describedherein to adopt it to various usages and conditions. Such embodimentsare also within the scope of the following claims.

[0318] All publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

1 68 1 20 DNA Artificial Sequence Synthetic Primer 1 agtgagataagcagtggatg 20 2 20 DNA Artificial Sequence Synthetic Primer 2 cttgtgctactcccatcact 20 3 22 DNA Artificial Sequence Synthetic Primer 3 ggagaccaccaaaccctcca aa 22 4 23 DNA Artificial Sequence Synthetic Primer 4gagagttgca gaaggggtyg act 23 5 20 DNA Artificial Sequence SyntheticPrimer 5 accacctatg acagcgtgac 20 6 20 DNA Artificial Sequence SyntheticPrimer 6 gtggcagcaa gtagacatcg 20 7 22 DNA Artificial Sequence SyntheticPrimer 7 gtcatcatct ctgccccttc tg 22 8 22 DNA Artificial SequenceSynthetic Primer 8 aacaacttct tgatgtcatc at 22 9 20 DNA ArtificialSequence Synthetic Primer 9 ccctcctctt tgtgctgtca 20 10 20 DNAArtificial Sequence Synthetic Primer 10 caccgtgctc tcatcggatg 20 11 23DNA Artificial Sequence Synthetic Primer 11 caggtgcagc tggtggagtc tgg 2312 20 DNA Artificial Sequence Synthetic Primer 12 caggagaaag tgatggagtc20 13 22 DNA Artificial Sequence Synthetic Primer 13 ggagaccaccaaaccctcca aa 22 14 20 DNA Artificial Sequence Synthetic Primer 14gtggcagcaa gtagacatcg 20 15 20 DNA Artificial Sequence Synthetic Primer15 caggagaaag tgatggagtc 20 16 20 DNA Artificial Sequence SyntheticPrimer 16 aggcagccaa cggccacgct 20 17 23 DNA Artificial SequenceSynthetic Primer 17 caggtgcagc tggtggagtc tgg 23 18 20 DNA ArtificialSequence Synthetic Primer 18 agtgagataa gcagtggatg 20 19 20 DNAArtificial Sequence Synthetic Primer 19 cttgtgctac tcccatcact 20 20 22DNA Artificial Sequence Synthetic Primer 20 ggagaccacc aaaccctcca aa 2221 23 DNA Artificial Sequence Synthetic Primer 21 gagagttgca gaaggggtygact 23 22 22 DNA Artificial Sequence Synthetic Primer 22 ggagaccaccaaaccctcca aa 22 23 22 DNA Artificial Sequence Synthetic Primer 23gagagttgca gaaggggtga ct 22 24 23 DNA Artificial Sequence SyntheticPrimer 24 caggtgcagc tggtggagtc tgg 23 25 20 DNA Artificial SequenceSynthetic Primer 25 caggagaaag tgatggagtc 20 26 22 DNA ArtificialSequence Synthetic Primer 26 gtcatcatct ctgccccttc tg 22 27 22 DNAArtificial Sequence Synthetic Primer 27 aacaacttct tgatgtcatc at 22 2829 DNA Artificial Sequence Synthetic Primer 28 gggaattcgg gtagaagttcactgatcag 29 29 28 DNA Artificial Sequence Synthetic Primer 29gggaattcgg gtagaagtca cttatgag 28 30 28 DNA Artificial SequenceSynthetic Primer 30 gggaattcgg gtagaagtca cttacgag 28 31 29 DNAArtificial Sequence Synthetic Primer 31 cccccaagct trcckgstyy cctctcctc29 32 29 DNA Artificial Sequence Synthetic Primer 32 cccccaagcttgcctggacc cctctctgg 29 33 32 DNA Artificial Sequence Synthetic Primer33 atcggcaaag cttggacccc tctctggctc ac 32 34 29 DNA Artificial SequenceSynthetic Primer 34 cccccaagct tctcggcgtc cttgcttac 29 35 29 DNAArtificial Sequence Synthetic Primer 35 gggaattcgg gtagaagttc actgatcag29 36 28 DNA Artificial Sequence Synthetic Primer 36 gggaattcgggtagaagtca cttatgag 28 37 28 DNA Artificial Sequence Synthetic Primer 37gggaattcgg gtagaagtca cttacgag 28 38 29 DNA Artificial SequenceSynthetic Primer 38 cccccaagct trcckgstyy cctctcctc 29 39 28 DNAArtificial Sequence Synthetic Primer 39 gggaattcgg gtagaagtca ctgatcag28 40 28 DNA Artificial Sequence Synthetic Primer 40 gggaattcgggtagaagtca cttatgag 28 41 28 DNA Artificial Sequence Synthetic Primer 41gggaattcgg gtagaagtca cttacgag 28 42 30 DNA Artificial SequenceSynthetic Primer 42 cttgaagacg aaagggcctc gtgatacgcc 30 43 30 DNAArtificial Sequence Synthetic Primer 43 ctgagacttc ctttcaccct ccaggcaccg30 44 31 DNA Artificial Sequence Synthetic Primer 44 cgatgaatgccccatttcac ccaagtctgt c 31 45 23 DNA Artificial Sequence SyntheticPrimer 45 ctgagccaag cagtggcccc gag 23 46 26 DNA Artificial SequenceSynthetic Primer 46 gggctgagac tgggtgaaca gaaggg 26 47 1479 DNA Bovine47 ggtaccgaaa ggcggccctg aacattctgc agtgagggag ccgcactgag aaagctgctt 60catcgccggg agggagccag ccagctacga ttgtgagcac gctcacagtg cacacggcat 120gtgcacggtc tcagcttaac caccttgaag gagtaactca ttaaagagcg tacgaatgca 180ttgataaaat gcacctgaga caaattaatt tcttaaacat cgactttgaa aatgaatata 240agtgagcagt tgataggctc tgaatgaaat accttccaac aggtgctgag aaccgccagg 300agcagggaac ggactccccg tggagcccca gaaggagcca gccctgatga tacctcggcc 360ctgggccctc ctcacgctgg gagagagcca gctcctgttg ttcatgcctg gcctgtggtt 420ctttgtcgtc atggccctca aacaagccca caggtcctgg cctgagtccc tcggcctgcg 480tgcagccgcc ccctcccctg ctggaggcac cctgcctgcc gtggagcccc tcacccaacg 540ttcccccgcc tgatgggttg ggccgcaaag gacaccgttt aaccagaact gccttccagg 600agcctactgc tgggaggcgg ccttctctgg gaccaggtcc actccactcc cttggatagt 660cactgtcagg cccctggtgg ccccacaaga ggcgtcctgg gaagccccag tctccttcca 720gcccctgaaa ttgcctccct ggagagccag atcaccctca cccagctccc tcccctggcc 780cccagggtct cctctcccat cccaccgccc accctaccct ggcgttgccg tcacagctaa 840cctgacctcc ctgggttcga gcgtgccgcc gcccctgtcg gcccccacct ggacccccgc 900agcctatctc tgagggctaa tgcccctgtc ccctgccccg ctgccagctg ccccctcttt 960ccaggccttt cctccgtgcc tctccagtcc tgcacctccc tgcagcttca cctgagactt 1020cctttcaccc tccaggcacc gtcttctggc ctgcaggtga ggtctcgcgc tccctcaggg 1080cacgatgtgg ctgcacacac accggccctc ctcccgagtc cctcctgcac acaccacgcg 1140cacccgaggt tgacaagccc tgccgtggtt gggattccgg gaatggcggc agagaggggc 1200ggggtgtcct tggggctggt ggcagggtcc tcatggatgc acacagcggc cccggctcag 1260gccaccttgg gaaaccagtc ctgggatctg caactcggcc atgttcctgc atctggacca 1320gccccaagac accaccccgg cgtggcgcca ctggcctggg aggagacaca tgtccctttc 1380ccatcagcaa tgggttcagc actaggatat gcagcacaca ggagtgtggc ttgggggtaa 1440aaaaaccttc acgaggaagc ggtttcacaa aataaagta 1479 48 3120 DNA Bovinemisc_feature (1)...(3120) n=a, t, c, g or no nucleotide 48 tctagacccaccagcctcag ttgaggttaa atggacccaa agcatctcaa caatttgccc 60 aagtcaagccagctcaatgg gttcccttct gttcacccag tctcagccca ccatggtaac 120 ccagcataccccggttaagc ccaggctagc ccagcccagc tgagcccagc tcagctcagt 180 tcagcccagttcaatccaga tcagcccaat ccaggccagc tcatcgagct cagttcagct 240 cagctcaaccctctcagccc agctcacctg ctcagccaag ctaagcccag ttcagcccag 300 ctcagcttaacccagctcac ccactctgcc cagctcagcc cagccctgct caactcagcc 360 cagcacagcccaacttggct cagctcagct tagcccagct cagcccagct tacccactcc 420 gcccagctcaaacagcccag gtcagcccaa cctagctcag ttcagcccag ctcagcccag 480 cccagctcagcccagctcac ccactctgcc cagctcaaca cagcccagct caacccagct 540 cagctcagttcagcccagct cacccactct gcccagctca ggccagctca acccagccca 600 gcccagctcactcattctgc caagctcagc ccagctcaac caggctcagc tcagctcagc 660 tcagccctgctgaccnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 720 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 780 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 840 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 900 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 960 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1020 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1080 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1140 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1200 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1260 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1320 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1380 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1440 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1500 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1560 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1620 nnnnngctcagctcagccca gctcagccca gcccagccca gctcacacac ttggcccacc 1680 tcagccactccattcagctc agcccagctc aacccagctc agctcagctc aacctagctc 1740 agccaagctaacccactcca cccagctcag cccagctcgc ccactctgcc cagctcaacc 1800 cagctcagctcagcccagcc cagyccagcc cagctcaccc actccatcca gcccagccca 1860 gcccagctgagcccagctca actcagccta acccagctca gcccagccta acccagctca 1920 gcccagcccaaccagctagc tgagcccagc tcagtgcagc tcaacccagc tcagctcagc 1980 tagcccagcccagctcaacc tggctcaacc cggctcagcc cagctcacct gctgtaggtg 2040 gcctgaaccgcgaacacaga catgaaagcc cagtggttct gacgagaaag ggtcagatcc 2100 tggaccatggccacggctaa aggccctggt ctgtggacac tgcccagctg ggctcatccc 2160 tcccagcctcttcccgcttc tcctcctggg agcccgctcg ccccttcccc tggtgcctga 2220 cacctccatcccgacaccag gcccagctgg cccttctccc agctgtcagt caccactacc 2280 ctccactctgggtgaaaagc ttgttggaga ctttagcttc cctagagcat ctcacaggct 2340 gagacacacttgccaccctc agagagaggc cctgtctctg ctgagcaggc agcgctgctt 2400 ctctgggagaggagagcctg ggcacacgtc cctgggtcct ggcctcctgg gcacgtgcca 2460 tgggcctgagatcccgcccc gagtctaaaa gagtcctggt gactaactgc tctctggcaa 2520 atgtcctcattaaaaaccac aggaaatgca tcttatctga acctgctccc aattctgtct 2580 ttatcacaaagttctgctga gaaagaggat actctctagc acagagacca tctgaacccc 2640 aaagctgcattgaacaccta agtgtggacg caggaagtgg tccctgtggg tgtgaagcac 2700 cccggcatcgcaggcagtag gtaaagacag attccctttc aagtagaaac aaaaacaact 2760 catacaaacatccctgggca gtgagtctgg ctgcaccggc tcctggtccc tggcatgtcc 2820 cctgggctctctgacctggg cggattcctc cgaatccctt cgctgtgtta actcgtgacc 2880 tgcctactggcctgggggca gaggccaggc ccacacgtcc ccaggtgtgg gcagtcccag 2940 gagaccccccagccttggcg agcctgggga ctcagagcag agactgtccc tccagacggt 3000 cccaggccccgctgactgcc gccccaccgg gcatcctctc aatcccccag ctagtagtgt 3060 agcagagtaactcacgacga atgcccccgt ttcacccaag tctgtcctga gatgggtacc 3120 49 146 DNABovine misc_feature (1)...(146) n=a,t,c, or g 49 gggaaggaag tcctgtgcgaccanccaacg gccacgctgc tcgtatccga cggggaattc 60 tcacaggaga cgagggggaaaagggttggg gcggatgcac tccctgagga gacggtgacc 120 agggttccnt ggccccagnngtcaaa 146 50 167 DNA Bovine 50 tttgactact ggggccaggg aaccctggtcaccgtctcct cagggagtgc atccgcccca 60 acccttttcc ccctcgtctc ctgtgagaattccccgtcgg atacgagcag cgtggccgtt 120 ggctgcctcg cacaggactt ccttcccgactccatcactt tctcctg 167 51 147 DNA Bovine misc_feature (1)...(147) n=a,t, c, or g 51 tttgacnnct ggggccangg aaccctggtc accgtctcct cagggagtgcatccgcccca 60 acccttttcc ccctcgtctc ctgtgagaat tccccgtcgg atacgagcagcgtggccgtt 120 ggntgcgtcg cacaggactt ccttccc 147 52 393 DNA Bovine 52ggaggcttgg tcaagcctgg agggtccctg agactctcct gtgcagcctc tggattcacc 60ttcagtgact actacatgag ctggatccgc caggctccag ggaaggggct ggagtgggtt 120tcatacatta gtagtagtgg tagtaccata tactacgcag actctgtgaa gggccgattc 180accatctcca gggacaacgc caagaactca ctgtatctgc aaatgaacag cctgagagcc 240gaggacacgg ctgtgtatta ctgtgcgaga ataactgggg atgcttttga tatctggggc 300caagggacaa tggtcaccgt ctcttcaggg agtgcatccg ccccaaccct tttccccctc 360gtctcctgtg agaattcccc gtcggatacg agc 393 53 131 PRT Bovine 53 Gly GlyLeu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 1 5 10 15 SerGly Phe Thr Phe Ser Asp Tyr Tyr Met Ser Trp Ile Arg Gln Ala 20 25 30 ProGly Lys Gly Leu Glu Trp Val Ser Tyr Ile Ser Ser Ser Gly Ser 35 40 45 ThrIle Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 50 55 60 AspAsn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala 65 70 75 80Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ile Thr Gly Asp Ala Phe 85 90 95Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Ser Ala 100 105110 Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn Ser Pro Ser 115120 125 Asp Thr Ser 130 54 411 DNA Bovine 54 gtggagtctg ggggaggcttggtacagcct gggaggtccc tgagactctc ctgtgcagcg 60 tcaggattca ccttcaggaactttggcatg cactgggtcc gccaggctcc aggcaagggg 120 ctggagtggg tgacagttatatggtatgac ggaagtaatc aatactatat agactccgtg 180 aagggccgat tcaccatctccagagacaat tccaagaaca tgttgtatct gcaaatgaac 240 agcctgagag ccgaggatacggctgtgtat tactgtgcga gagatcgcaa tggcctgaag 300 tacttcgatc tctggggccgtggcaccctg gtcactgtct catcagggag tgcatccgcc 360 ccaacccttt tccccctcgtctcctgtgag aattccccgt cggatacgag c 411 55 137 PRT Bovine 55 Val Glu SerGly Gly Gly Leu Val Gln Pro Gly Arg Ser Leu Arg Leu 1 5 10 15 Ser CysAla Ala Ser Gly Phe Thr Phe Arg Asn Phe Gly Met His Trp 20 25 30 Val ArgGln Ala Pro Gly Lys Gly Leu Glu Trp Val Thr Val Ile Trp 35 40 45 Tyr AspGly Ser Asn Gln Tyr Tyr Ile Asp Ser Val Lys Gly Arg Phe 50 55 60 Thr IleSer Arg Asp Asn Ser Lys Asn Met Leu Tyr Leu Gln Met Asn 65 70 75 80 SerLeu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg 85 90 95 AsnGly Leu Lys Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr 100 105 110Val Ser Ser Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser 115 120125 Cys Glu Asn Ser Pro Ser Asp Thr Ser 130 135 56 441 DNA Bovine 56accctcctca ctcactgtgc agggtcctgg gcccagtctg tgctgactca gccaccctca 60gcgtctggga cccccgggca gagggtcacc atctcttgtt ctggaagcag ctccaacatc 120ggaagtaatt atgtatactg gtaccagcag ctcccaggaa cggcccccaa actcctcatc 180tataggaata atcagcggcc ctcaggggtc cctgaccgat tctctggctc caagtctggc 240acctcagcct ccctggccat cagtgggctc cggtccgagg atgaggctga ttattactgt 300gcagcatggg atgacagcct gagtggtctt ttcggcggag ggaccaagct gaccgtccta 360ggtcagccca aggctgcccc ctcggtcact ctgttcccac cctcctctga ggagcttcaa 420gccaacaagg ccacactggt g 441 57 147 PRT Bovine 57 Thr Leu Leu Thr His CysAla Gly Ser Trp Ala Gln Ser Val Leu Thr 1 5 10 15 Gln Pro Pro Ser AlaSer Gly Thr Pro Gly Gln Arg Val Thr Ile Ser 20 25 30 Cys Ser Gly Ser SerSer Asn Ile Gly Ser Asn Tyr Val Tyr Trp Tyr 35 40 45 Gln Gln Leu Pro GlyThr Ala Pro Lys Leu Leu Ile Tyr Arg Asn Asn 50 55 60 Gln Arg Pro Ser GlyVal Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly 65 70 75 80 Thr Ser Ala SerLeu Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala 85 90 95 Asp Tyr Tyr CysAla Ala Trp Asp Asp Ser Leu Ser Gly Leu Phe Gly 100 105 110 Gly Gly ThrLys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser 115 120 125 Val ThrLeu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala 130 135 140 ThrLeu Val 145 58 459 DNA Bovine 58 agttggaccc ctctctggct cactctcttcactctttgca taggttctgt ggtttcttct 60 gagctgactc aggaccctgc tgtgtctgtggccttgggac agacagtcag gatcacatgc 120 caaggagaca gcctcagaag ctattatgcaagctggtacc agcagaagcc aggacaagcc 180 cctgtacttg tcatctatgg taaaaacaaccggccctcag ggatcccaga ccgattctct 240 ggctccagct caggaaacac agcttccttgaccatcactg gggctcaggc ggaggatgag 300 gctgactatt actgtaactc ccgggacagcagtggtaacc atgtggtatt cggcggaggg 360 accaagctga ccgtcctagg tcagcccaaggctgccccct cggtcactct gttcccaccc 420 tcctctgagg agcttcaagc caacaaggccacactggtg 459 59 153 PRT Bovine 59 Ser Trp Thr Pro Leu Trp Leu Thr LeuPhe Thr Leu Cys Ile Gly Ser 1 5 10 15 Val Val Ser Ser Glu Leu Thr GlnAsp Pro Ala Val Ser Val Ala Leu 20 25 30 Gly Gln Thr Val Arg Ile Thr CysGln Gly Asp Ser Leu Arg Ser Tyr 35 40 45 Tyr Ala Ser Trp Tyr Gln Gln LysPro Gly Gln Ala Pro Val Leu Val 50 55 60 Ile Tyr Gly Lys Asn Asn Arg ProSer Gly Ile Pro Asp Arg Phe Ser 65 70 75 80 Gly Ser Ser Ser Gly Asn ThrAla Ser Leu Thr Ile Thr Gly Ala Gln 85 90 95 Ala Glu Asp Glu Ala Asp TyrTyr Cys Asn Ser Arg Asp Ser Ser Gly 100 105 110 Asn His Val Val Phe GlyGly Gly Thr Lys Leu Thr Val Leu Gly Gln 115 120 125 Pro Lys Ala Ala ProSer Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 130 135 140 Leu Gln Ala AsnLys Ala Thr Leu Val 145 150 60 723 DNA Bovine 60 atgagattcc ctgctcagctcctggggctc ctcctgctct gggtcccagg atccagtggg 60 gatgttgtgc tgacccagactcccctctcc ctgtctatca tccctggaga gacggtctcc 120 atctcctgca agtctactcagagtctgaaa tatagtgatg gaaaaaccta tttgtactgg 180 cttcaacata aaccaggccaatcaccacag cttttgatct atgctgtttc cagccgttac 240 actggggtcc cagacaggttcactggcagt gggtcagaaa cagatttcac acttacgatc 300 aacagtgtgc aggctgaggatgttggagtc tattactgtc ttcaaacaac atatgtccca 360 aatactttcg gccaaggaaccaaggtagag atcaaaaggt ctgatgctga gccatccgtc 420 ttcctcttca aaccatctgatgagcagctg aagaccggaa ctgtctctgt cgtgtgcttg 480 gtgaatgatt tctaccccaaagatatcaat gtcaagtgga aagtggatgg ggttactcag 540 agcagcagca acttccaaaacagtttcaca gaccaggaca gcaagaaaag cacctacagc 600 ctcagcagca tcctgacactgcccagctca gagtaccaaa gccatgacgc ctatacgtgt 660 gaggtcagcc acaagagcctgactaccacc ctcgtcaaga gcttcagtaa gaacgagtgt 720 tag 723 61 20 DNAArtificial Sequence Synthetic Primer 61 gatgatgtct ccaggatgcc 20 62 21DNA Artificial Sequence Synthetic Primer 62 gacaagctta atatccgcag g 2163 30 DNA Artificial Sequence Synthetic Primer 63 aagaagagaa aggtagaagaccccaaggac 30 64 28 DNA Artificial Sequence Synthetic Primer 64cctgggtata gacaggtggg tattgtgc 28 65 40 DNA Artificial SequenceSynthetic Primer 65 ggggtctaga gcagacacta cactgatggg cccttggtcc 40 66 36DNA Artificial Sequence Synthetic Primer 66 ggggaagctt cgtgtccctggtcctgtctg acacag 36 67 34 DNA Artificial Sequence Synthetic Primer 67ggggctcgag gtcggcgaag gatgggggga ggtg 34 68 35 DNA Artificial SequenceSynthetic Primer 68 ggggggtacc gctgggctga gctgggcaga gtggg 35

1. A transgenic ungulate comprising one or more nucleic acids encodingall or part of a xenogenous immunoglobulin (Ig) gene which undergoesrearrangement and expresses more than one xenogenous Ig molecule.
 2. Theungulate of claim 1, wherein said xenogenous Ig molecule is a human Igmolecule.
 3. The ungulate of claim 1, wherein said nucleic acid iscontained within a chromosome fragment.
 4. The ungulate of claim 3,wherein said chromosome fragment is a ΔHAC or ΔΔHAC.
 5. The ungulate ofclaim 1, wherein said ungulate is a bovine, ovine, porcine, or caprine.6. A transgenic ungulate comprising a mutation that reduces theexpression of an endogenous antibody.
 7. The ungulate of claim 6,wherein said mutation reduces the expression of functional IgM heavychain or reduces the expression of functional Ig light chain.
 8. Theungulate of claim 6, comprising one or more nucleic acid encoding all orpart of a xenogenous Ig gene which undergoes rearrangement and expressesmore than one xenogenous Ig molecule.
 9. The ungulate of claim 6,wherein said ungulate is a bovine, ovine, porcine, or caprine.
 10. Anungulate somatic cell comprising one or more nucleic acids encoding allor part of a xenogenous Ig gene, wherein said gene is capable ofundergoing rearrangement and expressing one or more xenogenous Igmolecules in B cells.
 11. The cell of claim 10, wherein said nucleicacid encodes a xenogenous antibody.
 12. The cell of claim 10, whereinsaid nucleic acid is contained in a chromosome fragment.
 13. The cell ofclaim 10, wherein said cell is a fetal fibroblast or B-cell.
 14. Thecell of claim 10, wherein said ungulate is a bovine, ovine, porcine, orcaprine.
 15. An ungulate somatic cell comprising a mutation in a nucleicacid encoding an Ig heavy and/or light chain.
 16. The cell of claim 15,comprising a mutation in both alleles of said IgM heavy chain or saidlight chain.
 17. The cell of claim 15, further comprising one or morenucleic acids encoding all or part of xenogenous Ig gene, wherein saidgene is capable of undergoing rearrangement and expressing one or morexenogenous Ig molecules in B cells.
 18. The cell of claim 15, whereinsaid cell is a fetal fibroblast or a B-cell.
 19. The cell of claim 15,wherein said ungulate is a bovine, ovine, porcine, or caprine.
 20. Ahybridoma formed from the fusion of the B-cell of claim 13 or 18 with amyeloma cell.
 21. A method of producing antibodies, said methodcomprising the steps of: (a) administering one or more antigens ofinterest to an ungulate comprising nucleic acid encoding a xenogenousantibody gene locus, wherein the nucleic acid segments in said genelocus undergo rearrangement resulting in the production of antibodiesspecific for said antigen; and (b) recovering said antibodies from saidungulate.
 22. The method of claim 21, wherein said ungulate comprises amutation that reduces the expression of an endogenous antibody.
 23. Themethod of claim 21, wherein said nucleic acid is contained in achromosome fragment.
 24. The method of claim 21, wherein said nucleicacid is a human nucleic acid.
 25. The method of claim 21, wherein saidungulate is a bovine, ovine, porcine, or caprine.
 26. A method ofproducing antibodies, said method comprising recovering xenogenousantibodies from an ungulate comprising nucleic acid encoding axenogenous antibody gene locus, wherein the nucleic acid segments insaid gene locus undergo rearrangement resulting in the production ofxenogenous antibodies.
 27. The method of claim 26, wherein said ungulatecomprises a mutation that reduces the expression of an endogenousantibody.
 28. The method of claim 26, wherein said nucleic acid iscontained in a chromosome fragment.
 29. The method of claim 26, whereinsaid ungulate is a bovine, ovine, porcine, or caprine.
 30. A method ofproducing a transgenic ungulate, said method comprising the steps of:(a) inserting a cell, a chromatin mass from a cell, or a nucleus from acell into an oocyte, wherein said cell comprises a first mutation in anendogenous antibody heavy chain and/or light chain nucleic acid; and (b)transferring said oocyte or an embryo formed from said oocyte into theuterus of a host ungulate under conditions that allow said oocyte orsaid embryo to develop into a fetus.
 31. The method of claim 30, farthercomprising the steps of: (c) isolating a cell from said embryo, saidfetus, or an offspring produced from said fetus; (d) introducing asecond mutation in an endogenous antibody heavy chain and/or light chainnucleic acid in said cell; (e) inserting said cell, a chromatin massfrom said cell, or a nucleus from said cell into an oocyte; and (f)transferring said oocyte or an embryo formed from said oocyte into theuterus of a host ungulate under conditions that allow said oocyte orsaid embryo to develop into a fetus.
 32. The method of claim 30, whereinsaid cell comprises one or more nucleic acids encoding all or part of axenogenous Ig gene, wherein said gene is capable of undergoingrearrangement and expressing one or more xenogenous Ig molecules in Bcells, and wherein said cell is inserted into said oocyte.
 33. Themethod of claim 30, wherein said ungulate is a bovine, ovine, porcine,or caprine.
 34. A method of producing a transgenic ungulate, said methodcomprising the steps of: (a) inserting a cell having one or morexenogenous nucleic acids into an oocyte; wherein said xenogenous nucleicacid encodes all or part of a xenogenous Ig gene; said gene capable ofundergoing rearrangement and expressing more than one xenogenous Igmolecule in B cells; and (b) transferring said oocyte or an embryoformed from said oocyte into the uterus of a host ungulate underconditions that allow said oocyte or said embryo to develop into afetus.
 35. The method of claim 34, wherein said immunogloblulin chain isexpressed in serum and/or milk.
 36. The method of claim 34, wherein saidnucleic acid is contained in a chromosome fragment.
 37. The method ofclaim 34, wherein said xenogenous antibody is a human antibody.
 38. Themethod of claim 34, wherein said ungulate is a bovine, ovine, porcine,or caprine.
 39. A method of producing a transgenic ungulate, said methodcomprising the steps of: (a) inserting a cell, a chromatin mass from acell, or a nucleus from a cell into an oocyte, wherein said cellcomprises a first mutation in an endogenous gene, wherein said gene isnot naturally expressed by said cell; and (b) transferring said oocyteor an embryo formed from said oocyte into the uterus of a host ungulateunder conditions that allow said oocyte or said embryo to develop into afetus.
 40. The method of claim 39, further comprising the steps of: (c)isolating a cell from said embryo, said fetus, or an offspring producedfrom said fetus; (d) introducing a second mutation in an endogenous genein said cell; (e) inserting said cell, a chromatin mass from said cell,or a nucleus from said cell into an oocyte,; and (f) transferring saidoocyte or an embryo formed from said oocyte into the uterus of a hostungulate under conditions that allow said oocyte or said embryo todevelop into a fetus.
 41. The method of claim 39, wherein said cell is afibroblast.
 42. The method of claim 39, wherein said gene encodes anantibody.
 43. The method of claim 39, wherein said gene encodesalpha-(1,3)-galactosyltransferase.
 44. The method of claim 39, whereinsaid gene encodes prion protein.
 45. The method of claim 39, whereinsaid gene encodes J chain.
 46. The method of claim 39, wherein said cellcomprises a nucleic acid encoding an exogenous J chain.
 47. Ungulateantiserum comprising polyclonal human immunoglobulins (Igs). 48.Ungulate milk comprising polyclonal human Igs.